Culture medium

Endocr Regul, 1999 Dec, 33(4), 155 - 60
Triiodothyronine stimulates 3beta-hydroxysteroid dehydrogenase activity in the porcine corpus luteum; Gregorasczuk EL et al.; OBJECTIVE: To study the mechanism of thyroid hormone action on the activity of 3beta-hydroxysteroid dehydrogenase in the porcine corpus luteum . METHODS: Pig ovaries were obtained from slaughterhouse animals . Luteal cells were isolated from mid-developing (5-7 days after ovulation) corpora lutea and incubated for 24 h with or without triiodothyronine . Trilostane, an inhibitor od 3beta-HSD that blocks the conversion of pregnenolone to progesterone, was added to the medium in doses of 0,0.1, 1, 10 and 100 micromol . Each treatment was performed in triplicate and each culture system was set up in triplicate . Progesterone concentrations in culture media were determined by radioimmunoassays . RESULTS: Trilostane in a dose of 100 microM significantly decreased the basal progesterone secretion from luteal cells by 26% (P<0.05) . However, such secretion was increased by triiodothyronine (T3) in a dose of 10(-9) M . In addition, in T3-treated cells dose dependent inhibitory effect of trilostane on progesterone secretion was observed . Control cultures grown in control medium revealed a relatively weak 3beta-HSD activity which, however, markedly increased after the addition of T3 to the culture medium . Trilostane remarkably decreased 3beta-HSD activity in T3-stimulated cells . CONCLUSION: It was found that T3 acts on luteal cell steroidogenesis via the activation of 3beta-hydrosysteroid dehydrogenase in these cells.

Appl Environ Microbiol, 2000 Mar, 66(3), 1066 - 76
Prevention of yeast spoilage in feed and food by the yeast mycocin HMK; Lowes KF et al.; The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts . We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger . Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media . A partial purification protocol was developed, and a comparison with native W . mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt . Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent . The onset of aerobic spoilage in mature maize silage was delayed by application of A . niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts . This helped maintain both higher lactic acid levels and a lower pH . In yoghurt spiked with dairy spoilage yeasts, A . niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts . The higher the yeast growth rate, the more effective the killing action of the mycocin . Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.

Endocrinology, 2000 Mar, 141(3), 953 - 8
Identification of the lipophilic factor produced by macrophages that stimulates steroidogenesis; Nes WD et al.; Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells . This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility . The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly . Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns . MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point . Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy . The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns . A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography . The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol . Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol . When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected . Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity . Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions . Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h . Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.

Mol Hum Reprod, 2000 Mar, 6(3), 246 - 51
Effects of lipopolysaccharide and cytokines on production of RANTES by cultured human endometrial stromal cells; Arima K et al.; RANTES (regulated upon activation, normal T cell expressed and secreted), which is a potent chemoattractant for eosinophils, lymphocytes, and monocytes, was recently detected in the human endometrium . The effects of modulators of endometrial function, including lipopolysaccharide (LPS), tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-4 and interferon-gamma (IFN-gamma), on the production of RANTES by endometrial stromal cells (ESC) were examined by an enzyme-linked immunosorbent assay and Northern blot analysis . The concentration of RANTES in the culture media of non-stimulated ESC was below the level of detection . The concentration of RANTES was increased by the addition of TNF-alpha, IL-1beta and LPS . IFN-gamma synergistically enhanced the TNF-alpha- and LPS-induced RANTES expression, but had no effect on the IL-1beta-induced RANTES expression . The TNF-alpha-induced production of RANTES by ESC was inhibited by IL-4 . The transcription of RANTES in ESC was also stimulated by TNF-alpha, IL-1beta and LPS in a dose-dependent manner . It is suggested that the LPS and cytokines secreted by the maternal decidual tissue and the developing embryo may regulate the production of RANTES by ESC . The modulation of RANTES concentration in the local environment may contribute to the pathophysiological processes of human reproduction by regulating the immunological reaction at the fetal-maternal interface.

J Reprod Fertil Suppl, 1999, 54, 461 - 75
Development of serum-free culture systems for the ruminant embryo and subsequent assessment of embryo viability; Gardner DK; The mammalian embryo undergoes considerable changes in its physiology and energy metabolism as it proceeds from the zygote to the blastocyst stage . Complete development of the mammalian zygote in vitro was restricted to a few strains of mice and their F1 hybrids for many years, as the ruminant embryo arrested development at the 8- to 16-cell stage . The introduction of co-culture of ruminant embryos with somatic cells in the mid-1980s helped to alleviate this in vitro induced arrest . However, such culture systems required the use of complex tissue culture media and serum . Serum has subsequently been shown to induce several abnormalities during embryo development in culture and has been associated with the production of offspring with significantly greater birth weights than normal, leading to both difficulties in pregnancy management and an unacceptable frequency of neonatal death . Resurgence of interest in mammalian embryo physiology has culminated in the formulation of defined embryo culture media, capable of supporting a high percentage of viable blastocyst development in vitro . Optimum embryo development in culture has been shown to take place not in one, but two or more media, each designed to cater for the changing requirements and metabolism of the embryo as it develops . The development of viability assays to identify those embryos with the highest developmental potential will further increase the efficiency of embryo transfer procedures . Assays based upon nutrient uptake and subsequent utilization make promising candidates.

Clin Exp Allergy, 2000 Mar, 30(3), 348 - 55
Interleukin-13 and tumour necrosis factor-alpha synergistically induce eotaxin production in human nasal fibroblasts; Terada N et al.; BACKGROUND: There is increasing evidence that eotaxin is a key mediator in the development of tissue eosinophilia . However, the mechanism involved in the production of eotaxin has yet to be clarified . Most recently, it has been shown that interleukin (IL) -4 induces eotaxin in dermal fibroblasts . A novel cytokine termed IL-13, which binds to the alpha-chain of the IL-4 receptor, shares many biological activities with IL-4 . It is known that fibroblasts express the IL-4 receptor and produce collagen type I upon stimulation with IL-4 . OBJECTIVE: We investigated whether IL-13, as well as IL-4, are able to induce eotaxin production in human nasal mucosal fibroblasts (HNMFs) . Furthermore, we investigated the effect of costimulation of IL-13 and TNFalpha on eotaxin production . METHODS: HNMFs, isolated from inferior nasal mucosa samples, were stimulated by various kind of cytokines for 1-36 h at 37 degrees C in 5% CO2 . The change in the expression of eotaxin mRNA was then evaluated by reverse transcriptase-polymerase chain reaction and the Southern blot analysis . The amount of eotaxin in the culture media was measured by ELISA . RESULTS: IL-13 as well as IL-4 dose-dependently induced eotaxin expression in HNMFs . Furthermore, IL-13 and TNFalpha synergistically induced eotaxin expression in HNMFs, while they hardly induced eotaxin expression in endothelial cells, epithelial cells or eosinophils . The synergy was observed when pre-incubation of HNMFs with IL-13 was followed by a stimulation with TNFalpha, or HNMFs were simultaneously stimulated with IL-13 and TNFalpha . CONCLUSION: These results strongly indicate that IL-13, as well as IL-4, may be important in eotaxin-mediated eosinophilic inflammation in nasal mucosa . In addition, in nasal mucosa, fibroblasts are the major cell source for eotaxin.

J Dent Res, 2000 Jan, 79(1), 77 - 84
The expression of MMP-8 in human odontoblasts and dental pulp cells is down-regulated by TGF-beta1; Palosaari H et al.; Recent findings show that matrix metalloproteinase-8 (MMP-8) is expressed, in addition to neutrophils, by human chondrocytes, cultured fibroblasts, and endothelial cells . We investigated the expression of MMP-8 in other human mesenchyme-derived cells, odontoblasts, and pulp tissue . Odontoblasts and pulp tissue were collected from extracted human teeth for MMP-8 mRNA analysis with reverse-transcription/polymerase chain-reaction (RT-PCR) and Southern blot . The expression, localization, and secretion of MMP-8 protein were studied with Western blot, immunohistochemistry, and immunofluorometric assay . The effect of TGF-beta1 (10 ng/mL) on the expression, secretion, and concentration of secreted MMP-8 was studied by odontoblast and pulp tissue culture methods (Tjaderhane et al., 1998a) . RT-PCR demonstrated MMP-8 mRNA expression in native and cultured odontoblasts and pulp tissue and cultured pulp fibroblasts, with a 522-bp transcript comparable with that of bone marrow cells . The specificity of PCR was confirmed with Southern blot . Western blot with MMP-8-specific antibody detected 65- and 50-kDa proteins in native samples, representing latent and active forms of mesenchymal-type MMP-8, and in the conditioned odontoblast culture media, 50-kDa protein was observed . TGF-beta down-regulated the MMP-8 mRNA and concentration of secreted protein in both cultures . Immunohistochemical staining detected MMP-8 in odontoblasts . These findings indicate that mesenchyme-derived cells of the dentin-pulp complex express, synthesize, and activate MMP-8, which may, in concert with odontoblast-derived gelatinases, participate in organization of dentin organic matrix prior to mineralization.

Clin Cancer Res, 2000 Feb, 6(2), 493 - 7
Enhanced GBX2 expression stimulates growth of human prostate cancer cells via transcriptional up-regulation of the interleukin 6 gene; Gao AC et al.; Previous studies demonstrated that the GBX2 homeobox gene is consistently overexpressed in cultured human prostate cancer cell lines . In this study, the human GBX2 cDNA was cloned and a quantitative reverse transcription-PCR method used to demonstrate that GBX2 mRNA expression is enhanced in approximately 70% of human prostate cancer tissues compared with normal human prostate tissues . Purified recombinant GBX2 protein binds specifically to an ATTA motif within the promoter of the interleukin 6 (IL-6) gene . Using an antisense approach, down-regulation of the expression of GBX2 correlated with decreased expression of IL-6 and an inhibition of tumorigenicity of PC3 human prostate cancer cells . In addition, in vitro growth of the antisense clones was partially restored by exogenous addition of recombinant IL-6 protein to the culture media . These data demonstrated that enhanced GBX2 expression results in a stimulation of malignant growth of prostate cancer cells and that part of this stimulation involves up-regulation in the transcription of the IL-6 gene.

Int J Infect Dis, 2000, 4(1), 46 - 50
Generation of reactive oxygen species and formation and membrane lipid peroxides in cells infected with Chlamydia trachomatis; Azenabor AA et al.; OBJECTIVES: Chlamydiae are obligate intracellular pathogens that cause many diseases for which the pathogenic mechanisms are largely unknown . Because reactive oxygen species (ROS) have been implicated in pathogenesis of many viral and bacterial infections, the authors assessed the release of ROS in selected host cells (monocytes, Sup-T1 cells, and Hep-2 cells) infected with Chlamydia trachomatis . METHODS: Infected cell cultures demonstrated a dramatic depletion of uric acid from culture media that was not seen in uninfected cultures . Reactive oxygen species generated in infected cultures were associated with the formation of lipid peroxides in host cell membrane . RESULTS: There was a significant increase in lipid peroxide levels in infected cells compared to uninfected controls . Ascorbic acid treatment of infected cell cultures reduced the formation of membrane lipid peroxides . CONCLUSIONS: These results suggest that ROS produced during chlamydial replication cause membrane lipid peroxidation . The role of ROS-induced membrane damage in chlamydial pathogenesis is discussed.

Thromb Res, 2000 Jan 15, 97(2), 51 - 67
Recombinant human factor X: high yield expression and the role of furin in proteolytic maturation in vivo and in vitro; Himmelspach M et al.; Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FVIII and FIX inhibitor patients . This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 microg/mL x day and 78 microg/10(6) cells x day, that is to 100-fold higher levels than reported previously . Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-dependent gamma-carboxylation became impaired at these expression levels, up to 25% of the recombinant human factor X produced was active . This represents the highest functional activity ever reported for a vitamin K-dependent protein at such an expression level . Expression of recombinant human factor X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished . This suggests that a protease different from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors . In contrast, exposure of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a typical argP4-xP3-lys/argP2-argP1 Furin cleavage site in the propeptide, indicating relaxed specificity in vitro . Concomitantly with the degree of processing, the functional activity of recombinant human factor X increased . Interestingly, Furin was shown to even perform correct N-terminal proteolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro . Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor X light chain isoforms were observed and their structure characterized . One of these light chain forms correlated with the functional activity . Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal.

Biol Reprod, 2000 Mar, 62(3), 540 - 6
Platelet-activating factor induces an imbalance between matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 expression in human uterine cervical fibroblasts; Sugano T et al.; Platelet-activating factor (PAF) is involved in such reproductive processes as parturition . We investigated the effect of PAF on the expression of matrix metalloproteinase-1 (MMP-1) and that of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human uterine cervical fibroblasts . Uterine cervical tissue was obtained from patients who underwent cesarean section at term . Collagenase-dispersed fibroblasts were cultured and used in the experiments . PAF receptor was identified in the uterine cervical fibroblasts by use of reverse transcription-polymerase chain reaction and Southern blot analysis . Northern blot analysis showed that PAF increased the expression of MMP-1 mRNA in a time-dependent manner, whereas expression of TIMP-1 mRNA was not affected by PAF . Concentration of MMP-1 protein in the PAF-treated culture media significantly exceeded that in control cultures . The PAF-induced production of MMP-1 protein was abolished by treatment with WEB 2170, a specific PAF receptor antagonist . Results suggest that PAF may accelerate collagenolysis in the human uterine cervix by inducing an imbalance in the activity between MMP-1 and TIMP-1, thus contributing to the cervical ripening during parturition.

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Sep, 29(3), 259 - 63
{Application of mouse limb bud culture to study the influence of zinc on teratogenesis induced by cadmium}; Li Y et al.; In this study, an in vitro method of mouse limb bud culture in self-made rotator with continuous supplementation of gas mixture was employed in studying the teratogenic potential of cadmium and the influence of zinc on the teratogenesis induced by cadmium . Image analysis on the area and the form of the bone analgen of the cultured limb was used to evaluate quantitatively their teratogenic potentials . Different amounts of cadmium were directly added to culture medium . As cadmium concentrations were increased from 0.1 to 1.0 microgram/ml, the degree of morphogenetic differentiation and the area of the bone anlagen of limbs culture were significantly decreased . The paws and long bones were affected seriously . Cadmium had a greater effect on chondrogenic tissue than on soft tissue . Then various levels of Zn, together with cadmium (1.0 microgram/ml medium), were added into the culture media . As Zn concentrations increased from 1.0 to 10.0 micrograms/ml, the degree of morphogenetic differentiation and the area of cartilaginous bone anlagen of limbs culture were improved or increased . The long bones were better ameliorated as compared with the paw.

J Virol Methods, 2000 Feb, 84(2), 209 - 15
cDNA probes for detection of specific dsRNAs from the fungal pathogen, Monosporascus cannonballus; Batten JS et al.; Monosporascus cannonballus is an ascomycete fungus that is the causative agent of Monosporascus root rot/vine decline, a serious disease of muskmelon and watermelon . Double-stranded RNA (dsRNA) was identified in approximately 60% of M . cannonballus isolates recovered from infected muskmelon plants in 1993 . After repeated laboratory transfer on culture media, the majority of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to muskmelon . Initially, dsRNA purification and cDNA synthesis were attempted in three M . cannonballus isolates harboring dsRNAs . However, numerous difficulties were encountered due to the stable, double-stranded nature of the dsRNAs and contamination of the preparations by fungal rRNA . Several purification and cDNA protocols were evaluated and eventually modified into methods that were ultimately highly effective for cloning dsRNAs from M . cannonballus . The cDNAs derived from purified dsRNA preparations were cloned into a pUC119 plasmid vector and amplified in Escherichia coli . Nine cDNA clones were identified that are specific for medium-sized (ca . 3 kbp) dsRNAs associated with M . cannonballus isolate Ca91-17(96+) . The methods used to make the cDNA clones of the dsRNAs in M . cannonballus may be useful for those working on fungal dsRNAs . In addition, these cDNAs may be useful for identifying dsRNAs associated with the hypovirulence phenotype.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 47 - 54
Multi-step purification strategy for RANTES wild-type and mutated analogues expressed in a baculovirus system; Comuzzi B et al.; RANTES (regulated on activation, normal T cell expressed and secreted), a C-C chemokine, is one of the major HIV-suppressive factors produced by CD8+ T cells . Wild-type RANTES and genetically modified analogues were expressed in a baculovirus system and purified from cell culture supernatants employing a multi-step strategy based on affinity and RP-HPLC . Quantification and purity control of the final proteins were carried out by capillary electrophoresis using the synthetic or the recombinant wild-type RANTES as a reference . The procedure here reported requires only three days to obtain 0.016-0.270 mg of the pure and characterised proteins, starting from 370-900 ml of culture media, and is suitable for the analysis of a large number of RANTES analogues.

Cancer Res, 2000 Feb 1, 60(3), 741 - 8
Androgen deprivation of the PC-310 {correction of prohormone convertase-310} human prostate cancer model system induces neuroendocrine differentiation; Jongsma J et al.; Neuroendocrine (NE) cells are androgen-independent cells and secrete growth-modulating neuropeptides via a regulated secretory pathway (RSP) . We studied NE differentiation after androgen withdrawal in the androgen-dependent prostate cancer xenograft PC-310 . Expression patterns of chromogranin A, secretogranin III, and prohormone convertase-1 were analyzed at both protein and mRNA level to mark the kinetics of NE differentiation both in vivo and in vitro . PC-310 tumor-bearing nude mice were killed at 0, 2, 5, 7, 14, and 21 days postcastration . PC-310C cultures initiated from collagenase-treated tumor tissue could be maintained up to four passages, and androgen-deprivation experiments were performed similarly . PC-310 tumor volumes decreased by 50% in 10 days postcastration . Proliferative activity and prostate-specific antigen (PSA) serum levels decreased to zero postcastration, whereas PSA levels in PC-310C culture media first decreased and subsequently increased after 5 days . In vivo, androgen receptor (AR) expression decreased initially but returned to control level from 5 days postcastration on . CgA, secretogranin III, and secretogranin V expression increased in vivo from 5 days postcastration on . Subsequently, prohormone convertase-1 and peptidyl alpha-amidating monooxygenase as well as the vascular endothelial growth factor were expressed from 7 days postcastration on, and, finally, growth factors such as gastrin-releasing peptide and serotonin were expressed in a small part of the NE cells 21 days postcastration . The PC-310 tumors did not show colocalization of the AR on the NE cells in the tumor residues after 21 days . As in the PC-310 xenograft, NE differentiation was induced and AR expression relapsed after prolonged androgen suppression in PC-310C . For PC-310C cells, this relapse was associated with the secretion of PSA . PC-310C is the first culture of human prostatic cancer cells having the NE phenotype . The PC-310 model system is a potential androgen-dependent model for studying the role of NE cells in the progression of clinical prostate cancer . Androgen deprivation of NE-differentiated prostate cancer may induce the formation of both NE- and AR-positive dormant tumor residues, capable of actively producing NE growth factors via a RSP, possibly leading to hormone refractory disease.

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 924 - 7
Biological activity of human granulocyte colony stimulating factor with a modified C-terminus; Oshima Y et al.; Granulocyte colony-stimulating factor (G-CSF) undergoes receptor-mediated internalization into target cells which are normally restricted to neutrophilic granulocytes and their committed progenitor cells, suggesting that it may be applicable as a myeloid cell-targeting vehicle . To test this notion, we constructed a cDNA encoding a human G-CSF/murine stem cell factor (mSCF) chimeric molecule in a mammalian expression vector and transfected NIH3T3 cells with this plasmid . The resulting chimeric cytokine consisted of the entire G-CSF sequences fused to Lys148 of mSCF . It can be released from the surface membrane of NIH3T3 transformants through proteolytic cleavage at Ala164 of mSCF . The culture media conditioned by a number of stable transformants, which were confirmed by an enzyme-linked immunosorbent assay (ELISA) to secrete an hG-CSF derivative, were examined for their ability to stimulate CFU-G-derived colony formation as well as the proliferation of G-CSF-dependent NFS-60 cells . The results indicated that this C-terminus modified version of hG-CSF is as potent as recombinant hG-CSF in both assays .

Mol Cells, 1999 Dec 31, 9(6), 646 - 51
Effects of dopamine and melatonin on the regulation of the PIT-1 isotype, placental growth hormone and lactogen gene expressions in the rat placenta; Lee CK et al.; Rat placenta produces several members of the placental prolactin-growth hormone (PRL-GH), including placental lactogen (PL) and placental prolactin like protein (PLP), during pregnancy . It is important to study placental local regulators that control the expression of PRL-GH genes . We have previously reported that dopamine (DA) can regulate Pit-1 and PL-II gene expressions . In this study we aimed to investigate the local expression of melatonin receptor 1a (Mel1a) and the effects of DA and melatonin on the expressions of PL-Iv, PL-II, PLP-C genes and Pit-1 gene that are involved in the expression of PRL-GH genes in the rat pituitary and placenta . According to the Northern blot analysis, DA receptor 2 (D2) was expressed in the rat placenta . We also report on the local expression of Mel1a in the rat placenta for the first time . Injected DA agonist, bromocriptine (in vivo) decreased PL-Iv, PLP-C and Pit-1 mRNA levels in the rat placenta . The melatonin agonist, chloromelatonin in culture media also decreased the levels of PL-Iv, PL-II and PLP-C mRNA . However, melatonin does not affect the Pit-1 mRNA level . These data suggest that D2 and Mel1a may control the expression of PRL-GH genes in the rat placenta and its response to the extracellular changes of DA and melatonin secreted from the maternal organ . However, Pit-1 may not be involved in the Mel1a induced inhibition of PRL-GH gene expressions in the rat placenta.

Endocrine, 1999 Aug, 11(1), 31 - 6
Antisteroidogenic action of nitric oxide on human corpus luteum in vitro: mechanism of action; Johnson MC et al.; To analyze the mechanism by which nitric oxide (NO) exerts its antisteroidogenic action, human luteal cells were cultured during 24 and 48 h with L-arginine (L-Arg, 1 mmol/L); 1,2(2-trifluoromethylphenyl)imidazole (TRIM) (50 micromol/L and 1 mmol/L) and cyclic guanosine monophosphate (cGMP) analog (8-Br-cGMP, 1 mmol/L) . Estradiol, nitrite, and P450 AROM activity were determined in culture media . Total cGMP concentration was evaluated in the cells and culture media by radioimmunoassay, and NADPH diaphorase was used as a histochemical marker for NO synthase (NOS) activity . During the corpus luteum (CL) life-span, NO affected estradiol secretion in an age-dependent manner, with an inhibition in mid-CL (37%; p < 0.05) in agreement with our previous results, and no significant modification in early and late CL . Basal nitrite concentration in 24 and 48 h of midluteal cell cultures (42 and 93 pmol/10(6) cells, respectively) was increased by L-Arg (53% and 88%) and inhibited by the two TRIM concentrations; also, an intense diaphorase reactivity was observed in endothelial cells and luteal parenchyma . Total cGMP was not detected in cell cultures and 8-Br-cGMP did not modify estradiol secretion, whereas aromatase activity was strongly inhibited by L-Arg (70%, p < .05) . These results suggest that both NOS isoforms are active in midluteal cells, and the mechanism of action for NO on in vitro estradiol secretion may be an inhibition of P450 AROM activity.

Curr Opin Urol, 1999 Nov, 9(6), 541 - 5
Oocyte insemination with spermatozoa precursors; Schoysman R et al.; Since the use of testicular spermatozoa in programs of assisted fertilization proved very successful, attention was focussed on the use of spermatids also carrying 23 chromosomes . Several difficulties became obvious; the first one concerned the recognition of round spermatids . This is a problem which does not concern elongating and elongated cells . The intra-cytoplasmic injection of elongated spermatids resulted in several pregnancies but this is not so for the round ones . Although, in the group of patients in whom only round spermatids are found at the time of the attempt, is to be divided into two categories; patients in whom previous research allowed to find spermatozoa, however few, and patients who never produced spermatozoa at all . This last group is no longer an indication for intracytoplasmic sperm injection procedure unless in the future new culture media allow a maturation into elongated forms.

Medicina (B Aires), 1999, 59 Suppl 2, 57 - 62
{Myocardial cell response to Trypanosoma cruzi infection}; Postan M et al.; The aim of this study is to establish the response of cardiac myocytes to the infection with Trypanosoma cruzi . The role of myocardial cell proliferation on heart remodelation and the ability of these cells to produce nitric oxide and control intracellular parasite growth during T . cruzi infection were evaluated . The presence of proliferating cell nuclear antigen (PCNA) was determined in myocardial cells of Wistar rats infected with T . cruzi, resulting in a significant increase of PCNA+ labelling in all stages of disease . The ability of myocardial cells to control growth of intracellular parasites and the production of nitric oxide were evaluated in cultures of cardiac myocytes obtained from neonatal rats . Different combinations of cytokines were added to culture media . The number of cardiac cells displaying intracellular amastigotes was lower in cultures supplemented with IL-1b, TNF-a and IFN-g than with other cytokine combinations and controls . The addition of cytokines resulted also in an increase of nitric oxide production in both infected and non-infected controls . These results demonstrate that myocardial cells participate actively in the response of the heart to the infection with T . cruzi.

Pediatr Surg Int, 2000, 16(1-2), 91 - 3
The effects of sucralfate and selective intestinal decontamination on bacterial translocation; Akman M et al.; Sucralfate is widely used as a cytoprotective agent in patients with peptic ulcer and other intestinal mucosal damage . In this study, the effects of sucralfate and/or selective intestinal decontamination with gentamycin on bacterial translocation (BT) in rats with experimentally-induced mechanical jaundice were investigated . Seventy-five adult male Wistar albino rats were divided into five groups of 15 each . In all except a sham group, we performed ligation of the common bile duct (CBD) via a vertical laparatomy . After surgery, the rats in group 1 were treated with oral sucralfate (5 mg/kg per day); those in group 2 underwent oral gentamycin therapy (5 mg/kg per day) for 5 days . Group 3 rats were treated with sucralfate and gentamycin for 5 days subsequent to the operation . The rats in group 4 served as controls, and received only 0.9% saline solution . Group 5 was a sham group . After 5 days of surgery, all rats were killed; the mesenteric lymph nodes (MLN), liver, and a segment of terminal ileum were harvested aseptically . The collected tissues were cultured in McCaunkey medium and chocolate agar . For each specimen, the colony-forming units (CFU) were calculated and the percentage of viable translocated micro-organisms was counted . In all rats who had ligation of the CBD, high numbers of bacteria were demonstrated in the liver, MLN, and ileum . In the liver of rats with sucralfate and/or gentamycin treatment, there was a marked reduction in CFU compared to the control group . Similarly, in the MLN measurements of CFU were higher in the control rats than the study groups . In both McCaunkey and chocolate media, the numbers of bacteria in control rats were significantly higher than in the study groups (P < 0.001) . However, among the study groups themselves there was no significant difference in CFU in any of the specimens or culture media (P > 0.05) . Experimentally-induced mechanical jaundice from ligation of the CBD causes significant BT in rats . Sucralfate and/or gentamycin may reduce the degree of BT from the bowel mucosa . We did not find any difference in protection from BT between sucralfate and gentamycin or both in rats with experimentally-induced mechanical jaundice.

Ann Hematol, 2000 Jan, 79(1), 13 - 9
Ex vivo expansion of CD34+/CD41+ late progenitors from enriched peripheral blood CD34+ cells; Halle P et al.; In our experience, patients with neuroblastoma who undergo transplantation with CD34+ cells following high-dose chemotherapy have prolonged delays in platelet recovery . In vitro expansion of megakaryocyte (MK) cells may provide a complementary transplant product able to enhance platelet production in the recipient . We investigated the ability of a combination of various hematopoietic growth factors to generate ex vivo MK progenitors . Immunoselected CD34+ cells from peripheral blood stems cells (PBSCs) were cultured in media with or without serum, supplemented by IL-3, IL-6, IL-11, SCF, TPO, Flt-3 ligand, and MIP-1alpha . In terms of MK phenotypes, we observed a maximal expansion of CD61+, CD41+, and CD42a of 69-, 60-, and 69-fold, respectively, i.e., 8-10 times greater than the expansion of total cell numbers . Whereas the absolute increment of CD34+ cells was slightly elevated (fourfold) we showed increases of 163-, 212-, and 128-fold for CD34+/CD61+, CD34+/CD41+, and CD34+/CD42a+ cells, respectively . We obtained only a modest expansion of CFU-MKs after only 4 days of culture (fourfold) and similar levels of CFU-MKs were observed after 7 days (fivefold) . Morphology and immunohistochemistry CD41+ analyses confirmed expansion of a majority of CD41+ immature cells on days 4 and 7, while on day 10 mature cells began to appear . These results show that primarily MK progenitors are expanded after 4 days of culture, whereas MK precursor expansion occurs after 7 days . When we compared the two culture media (with and without serum) we observed that increases of all specific phenotypes of the MK lineage were more elevated in serum-free culture than in medium with serum . This difference was especially marked for CD34+/CD61+ and CD34+/CD41+ (163 vs 42 and 212 vs 36, respectively) . We contaminated CD34+ cells with a neuroblastoma cell line and we observed no expansion of malignant cells in our culture conditions (RT-PCR for tyrosine hydroxylase positive at day 4 and negative at day 7) . With our combination of hematopoietic growth factors we are able to sufficiently expand ex vivo MK late progenitor cells to be used as complementary transplant products in neuroblastoma patients who undergo transplantation with CD34+ cells . It is possible that these committed MK late progenitors could accelerate short-term platelet recovery in the recipient until more primitive progenitor cells have had time to engraft.

Toxicol Appl Pharmacol, 2000 Feb 15, 163(1), 60 - 6
Toxicity of heparin in postimplantation whole-embryo culture; Kesby GJ; Cranial neural tube defects occur when heparin is added to the culture media of postimplantation rat embryos undergoing organogenesis in vitro . Timed-exposure studies were undertaken to determine whether the defects caused by heparin were the result of defective folding and fusion of the neural folds, or due to reopening of a closed neural tube . Experiments were also undertaken to elucidate whether the in vitro toxicity of heparin was due to an effect of heparin at the level of the culture medium, at the level of the visceral yolk sac, or at the level of the embryo proper . Heparin was found to cause defective folding and closure of the neural folds at between 9.5 and 10.5 days' gestation . Neural tube defects did not occur when embryos were cultured in media prepared using a culture medium depleted of heparin ligands by heparin-agarose affinity chromatography . However, studies with {G-(3)H}heparin demonstrated visceral yolk sac uptake and transfer of the radiolabel to the embryo proper . In addition, microinjection of heparin directly into the amniotic cavity of early head-fold embryo explants resulted in cranial neural tube defects similar to those caused by the addition of heparin to culture medium . These data indicate that heparin causes closure defects of cranial neurulation, primarily by an effect at the tissue level of the embryo proper .

Biotechnol Prog, 2000 Jan-Feb, 16(1), 125 - 32
Cell-microcarrier adhesion to gas-liquid interfaces and foam; Bauer P et al.; The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied . These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces . Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer . These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces . Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells . No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces . It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium . It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.

Luminescence, 2000 Jan-Feb, 15(1), 45 - 9
Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein; Liu J et al.; We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP . In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells . Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein . Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy . Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity . This fusion protein could be a useful marker for the study of protein secretion in mammalian cells .

J Endocrinol, 2000 Feb, 164(2), 139 - 47
Evidence for complement-dependent and -independent inhibition of insulin secretion from clonal beta-cells incubated in the presence of sera of newly diagnosed IDDM patients; Conroy SJ et al.; There are conflicting reports on the effect of serum from patients with insulin-dependent diabetes mellitus (IDDM) or normal human serum on beta-cell function and insulin secretion . Here, we report that the sera of newly diagnosed IDDM patients potently suppresses insulin secretion from a clonal rat pancreatic beta-cell line (BRIN-BD11), but do not alter cell viability . Indeed, the viability of the beta-cells was not significantly different between cells cultured in 10% (v/v) IDDM sera, normal human sera, or fetal calf serum after 24, 48 and 72 h . Alanine-stimulated insulin secretion from cells cultured for 24 h in (10% v/v) IDDM patient sera was reduced to 48% of that secreted from cells cultured in (10% v/v) normal human sera . After depletion of the complement components C1q and C3, the inhibition of insulin secretion induced by IDDM patient sera was significantly reversed (no significant difference was observed between cells cultured in complement-depleted IDDM patient sera and cells cultured in normal human sera or complement-depleted normal human sera) . The concentration of glutamic acid decarboxylase (GAD) autoantibodies was markedly increased in the sera of six out of nine newly diagnosed IDDM patients in this study, whereas insulin auto-antibodies (IAA) were detected in the sera of three of the nine patients and islet-cell antibodies (ICA) in the sera of five of them . In addition, the concentration of soluble terminal complement complexes (SC5-9) was greater in some of the beta-cell culture media samples after 24 h incubation when the incubation medium was supplemented with IDDM patient sera than when supplementation was with normal human sera . We propose that the mechanism of sera-induced inhibition of insulin secretion from clonal beta-cells may involve complement- and cytokine-stimulated intracellular events that attenuate the metabolite-induced secretory process.

J Mol Endocrinol, 2000 Feb, 24(1), 135 - 44
Differential expression of 17beta-hydroxysteroid dehydrogenases types 2 and 4 in human endometrial epithelial cell lines; Husen B et al.; In the endometrium two enzymes are known to convert estradiol to its inactive metabolite estrone: microsomal 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) and peroxisomal 17beta-HSD4 . In order to elucidate the particular function of each of these two different enzymes, the human endometrial epithelial cell lines HEC-1-A and RL95-2 were examined with respect to the expression of 17betaHSD isozymes . They were compared with human endometrium in vivo . Non-radioactive in situ hybridization revealed both enzymes in glandular epithelial cells of human endometrium . The two cell lines were screened for mRNA expression of 17beta-HSD 1-4 by RT-PCR and Northern blot . 17beta-HSD2 and 4 could be detected by either method, 17beta-HSD1 only by RT-PCR, 17beta-HSD3 not at all . Both cell lines were proven to have no receptor for progesterone which is known as a physiological inducer of several 17beta-HSD isozymes . To study the regulation of 17beta-HSD2 and 17betaHSD4, the concentration of fetal calf serum in the cell culture media was reduced stepwise to 0.3% by dilution with a defined serum replacement . This treatment led to an inhibition of 17beta-HSD2 mRNA expression and an increase in the mRNA expression of 17beta-HSD4 . Concomitantly, distinct morphological changes were observed, such as a decrease in the number and length of microvilli and a decrease in the formation of domes on top of the monolayers . The endometrial epithelial cell lines HEC-1-A and RL95-2 represent a suitable in vitro model for further studies of the differential expression of the major endometrial HSD isozymes, independent of the effect of progesterone.

Hum Reprod, 2000 Feb, 15(2), 395 - 401
A single medium supports development of bovine embryos throughout maturation, fertilization and culture; Gandhi AP et al.; Oocytes and embryos are typically exposed sequentially to varying culture media in standard in-vitro protocols . Expenditures of energy may be required following each medium change to adjust to the changing environment . Therefore, a single base medium was evaluated for its ability to support in-vitro maturation, fertilization and pre-implantation development (IVM/F/C) of bovine oocytes and embryos . Four treatments were examined: a standard maturation {tissue culture medium (TCM) 199 with bovine calf serum (BCS)}, fertilization (modified Tyrode's medium with albumin, lactate and pyruvate) and culture (hamster embryo culture medium/TCM with BCS) system (control) and three synthetic oviductal fluid (SOF) treatments; maturation in SOF with bovine serum albumin (SOFBSA), SOF with bovine calf serum (SOFBCS) or the control maturation medium (TCM199 with BCS; SOF199), followed by fertilization and culture in SOF medium . The percentage of total inseminated oocytes successfully developing to the morula and blastocyst stage did not differ (P > 0 . 05) between treatments (control, 30.5 +/- 3.5; SOFBSA, 24.6 +/- 3.2; SOFBCS, 22.4 +/- 4.7; SOF199, 27.3 +/- 3.2) . Embryos cultured in SOFBCS (92.1 +/- 6.4) had significantly higher cell numbers (P < 0 . 05) than those cultured in control (74.8 +/- 4.8) and SOFBSA (71.6 +/- 6.6) but not SOF199 (81.2 +/- 6.8) . In conclusion, a single medium can be used successfully throughout maturation, fertilization and pre-implantation embryo development . Moreover, inclusion of serum during maturation in the single medium system resulted in significantly greater cell numbers, possibly reflecting increased quality of the embryos produced.

Anticancer Res, 1999 Jul-Aug, 19(4B), 2963 - 8
Production of amyloid beta protein precursor as a proteinase inhibitor by human astrocytic tumors; Nakagawa T et al.; The production of amyloid beta protein precursor (APP), which is a potent inhibitor of matrix metalloproteinases and serine proteinases, in human astrocytic tumors (n = 17) and normal brain tissues (n = 3) was investigated . We found proteinase inhibitory activity at around 120 kD by trypsin reverse zymography in the culture media of explant cultures of anaplastic astrocytomas and glioblastomas, but not in those of astrocytomas and normal brain tissues . Immunohistochemistry using a monoclonal antibody against human APP demonstrated that APP was detectable mainly in tumor and endothelial cells . Semiquantative analysis of western blotting revealed that immunoreactivity for APP in the culture media of tumor explant cultures appeared to be increased associated with the malignancy of astrocytic tumors . These findings suggest that APP production may be related to the malignant progression of human astrocytic tumors.

Biotechniques, 2000 Jan, 28(1), 124 - 6, 128-30
Cloning trap for signal peptide sequences; Lim SP et al.; Novel secreted and/or type I transmembrane proteins containing N-terminal signal sequences have been successfully cloned using the signal sequence trapping (SST) method . Often this involves random cloning of short 5' cDNA terminal ends into an epitope-tagged expression vector and the detection of expressed recombinant proteins on the cell surfaces of transfected cells with an antibody to the tagged epitope . Here, we report a novel cloning system for the detection of secreted proteins also using SST . In this method, we used the human immunodeficiency virus (HIV-1) p24 as the epitope for tagging . To test the system, two constructs were created . The 5' terminal end of a human beta-chemokine (which was regulated upon activation, expressed by normal T cells and presumably secreted {RANTES}) and the 5' end of a human CD4 receptor were cloned upstream of and in-frame with the p24 cDNA . Secreted p24 was detectable in the culture media two days after transfection of either DNA construct into the human cell lines, HeLa and 293T . When the chimeric p24 expression constructs were transfected at a ratio of 1:100 to the vector pcDNA3.1(+), p24 could still be detected in cell supernatants . The use of a secreted viral antigen like HIV-1 p24 (or of any noncellular protein) as a marker in SST cloning approaches is likely to be advantageous because it reduces the background noise in detection and also renders this system suitable for high-throughput screening.

Biochemistry (Mosc), 1999 Dec, 64(12), 1408 - 17
A sensitive assay of translational fidelity (readthrough and termination) in eukaryotic cells; Sogaard TM et al.; The process of translation termination in eukaryotes has been monitored by different types of assays, each with its own merits . We have developed an in vivo system where the reporter protein is secreted from the cells in culture thus enabling continuous monitoring of translation termination activity by simple sampling of the cell culture media . Using this system, cell cultures can be challenged with various stimuli during growth and the cellular responses on the translational level can be investigated in vivo as well as in vitro . Sampling is rapid, easy, and non-destructive to the cells, which enables measurement of translational fidelity in real time during time-course experiments . In particular with this system it is possible to assess very low levels of stop codon suppression . The reporter enzyme, secreted alkaline phosphatase (SEAP), becomes tagged with the S-peptide when there is readthrough of a stop codon placed between the C-terminus of the SEAP and the S-peptide . The tagged SEAP is bound to a matrix and the bound SEAP activity is measured versus total SEAP activity in the medium as a reference . With this assay we have confirmed that eRF1 acts as an antisuppressor in cells transfected with a cognate suppressor tRNA as well as in control cells, where a small but significant level of readthrough (suppression) could be detected . We have also characterized suppression of the three stop codons individually, and especially UGA is prone for wobbling.

Liver Transpl, 2000 Jan, 6(1), 76 - 84
Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes; Nyberg SL et al.; A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro . The PERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL) . We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes . Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL . Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested) . Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase . Primers targeting the pol gene of PERV were used for PCR . Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene . Levels of PERV sequences were estimated by serial dilution . All positive samples were tested for infectivity in HEK293 cells . Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively . Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA) . PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes . The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA . In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera) . These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.

Antisense Nucleic Acid Drug Dev, 1999 Dec, 9(6), 497 - 505
Delivery of antisense oligonucleotides and plasmid DNA with various carrier agents; Kang SH et al.; A series of cationic nucleic acid carriers was evaluated for their ability to deliver pLuc plasmid DNA or a 2'-O-methyl-oligoribonucleoside phosphorothioate, ON-705 . Oligonucleotide delivery and its antisense function were assayed by a recently developed assay based on alternative splicing of modified luciferase pre-mRNA (Kang et al., 1998) . This assay scores only the nuclear and sequence-specific antisense activity of the oligonucleotides . The results show that the efficiencies of delivery of plasmid DNA and oligonucleotides by the tested carriers, with the exception of Exgene and Lipofectamine, differed markedly . The efficiency of the delivery of ON-705 oligonucleotide was reduced by 70%-90% for all carriers, except Effectene, in culture media containing 8% fetal bovine serum . Interestingly, the efficiency of delivery of the ON-705-Effectene complex increased with serum concentrations of up to 30%.

Am J Nephrol, 2000 Jan-Feb, 20(1), 82 - 8
Pentoxifylline reduces in vitro renal myofibroblast proliferation and collagen secretion; Hewitson TD et al.; Interstitial myofibroblasts (MF) are cells with features of both smooth muscle cells and fibroblasts . They have been universally recognized in situations of tubulointerstitial injury, where their presence has been shown to be a marker of disease progression . The objective of this study was to determine if functions of MF relevant to fibrogenesis can be modified in vitro by the phosphodiesterase inhibitor pentoxifylline (PTX) . MF were obtained from sub-culture of normal rat kidney explant outgrowths maintained in DMEM + 20% fetal calf serum (FCS), supplemented with antibiotics . Cells were characterized on the basis of growth characteristics and immunohistochemistry . MF constituted >95% of cells at passage 3 . Cell culture media was supplemented with the potential antagonist PTX alone (0, 1, 10, 100 microg/ml) and in combination with TGFbeta(1) (5 ng/ml) . Population kinetics, proliferation and collagen production were determined from cell growth, {(3)H}thymidine incorporation and {(3)H}proline incorporation in collagenous proteins, respectively . Both serum-stimulated population growth and proliferation were reduced in a linear fashion by 1, 10 and 100 microg/ml PTX (all p < 0.05 versus 0 microg/ml) . Effect of PTX on cell population growth was however reversible when PTX was removed . Basal collagen secretion was decreased by PTX at 10 and 100 microg/ml (p < 0.05 versus 0 microg/ml although layer collagen remained unchanged . Collagen production (secreted and cell layer) was augmented by 5 ng/ml TGFbeta(1) . These effects on collagen production were partially reduced when 100 microg/ml PTX was added . The authors conclude that myofibroblast function can be altered with agonists/antagonists . Attempts to down-regulate fibrogenic functions of MF may therefore offer a valuable therapeutic strategy .

J Reprod Fertil Suppl, 1998, 53, 109 - 18
Maturation of redox regulatory mechanisms in the epididymis; Aitken RJ et al.; As spermatozoa pass through the epididymis they complete a maturation process that enables these cells to participate in the process of fertilization . Epididymal maturation involves a complex cascade of changes involving the remodelling of the sperm surface, the induction of chromatin condensation, the acquisition of movement, and development of the potential for capacitation . In this review we shall consider how changes in the redox status of mammalian spermatozoa may contribute to the completion of these maturation events . Spermatozoa from all regions of the epididymis exhibit a spontaneous capacity for superoxide anion production which can be enhanced by exposure to NADPH, particularly in the caput region . It is hypothesized that this spontaneous free radical generating activity is mediated by a membrane-bound NADPH oxidase, the function of which is to generate the peroxides that are needed to serve as hydrogen acceptors for phospholipid hydroperoxide glutathione peroxidase in the induction of sperm chromatin condensation . As spermatozoa enter the cauda epididymidis they also express a capacity for hydrogen peroxide (H2O2) generation when released into simple, defined culture media . The onset of this activity is thought to be associated with the induction of sperm capacitation through stimulation of the tyrosine phosphorylation events involved in the attainment of a capacitated state . It is concluded that sperm maturation is a dynamic, redox regulated process, any imbalance in which could lead to the production of spermatozoa that are compromised in terms of their potential for fertilization and the integrity of their DNA.

Neurosci Lett, 1999 Dec 17, 277(1), 33 - 6
Inhibition of prolylendopeptidase does not affect gamma-secretase processing of amyloid precursor protein in a human neuroblastoma cell line; Johnston JA et al.; Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease . The enzyme activities (beta- and gamma-secretases) involved in generating Abeta from amyloid precursor protein (APP) are unidentified . It has been suggested that prolylendopeptidase (PEP), an oligopeptidase that normally cleaves after proline residues, could also cleave after the alanine at position 42 of Abeta to generate Abeta42 . We investigated whether inhibition of PEP activity in human neuroblastoma cells affected Abeta levels in cell culture media . An SH-SY5Y cell line expressing SPA4CT, encoding the C-terminal 100 residues of APP and the signal sequence, was used . Only gamma-secretase activity is required for Abeta production in this cell line . The PEP inhibitor Fmoc-AlaPro-CN (10 microM) reduced PEP activity in these cells by approximately 95% in the absence of significant toxicity, but had no effect on Abeta40 or Abeta42 levels in cell culture media . We conclude that PEP is unlikely to be involved in gamma-secretase processing of APP.

J Food Prot, 2000 Jan, 63(1), 96 - 101
Utilization and transport of acetic acid in Dekkera anomala and their implications on the survival of the yeast in acidic environments; Geros H et al.; The yeast Dekkera anomala IGC 5153 exhibited a restricted ability to use weak acids as the only carbon and energy sources . Of the monocarboxylic, dicarboxylic, and tricarboxylic acids tested, only acetic acid was used in such a way . The cells were able to grow at acetic acid concentrations of 0.1 to 3% (vol/vol) over a pH range of 3.5 to 5.5, and the specific growth rates decreased exponentially with the increase of the undissociated acetic acid concentration in the culture medium . Transport assays carried out in cells that exhibited higher specific growth rates showed the presence of an acetate-proton symport associated with a simple diffusion component of the undissociated acetic acid, the weight of the latter increasing with the undissociated acid concentration in the culture media . The acetate carrier was shared by propionic, formic, and sorbic acids and was inducible and repressed by glucose and concentrations of undissociated acetic acid in the culture medium above 0.3% (vol/vol) . In undissociated acetic acid repression conditions, the lowest values for the yeast specific growth rates were obtained, and the simple diffusion of the undissociated acid was the only mechanism involved in the acetic acid uptake by the cells . The results will be discussed in terms of the high tolerance of D . anomala to the acidic stress conditions present in wine.

Biol Reprod, 2000 Feb, 62(2), 334 - 9
Expression of parathyroid hormone-related peptide (PTH-rp) and its receptorin the porcine ovary: regulation by transforming growth factor-beta and possible paracrine effects of granulosa cell PTH-rp secretion on theca cells; Garmey JC et al.; Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues . To evaluate the expression of this Ca(2+)-regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR) . The cDNA encoding PTH-rp (419 base pairs {bp}) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes . Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1-5 mm) and medium-sized (5-8 mm) antral follicles . In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells . Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells . Transforming growth factor (TGF)-beta1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h . TGF-beta1 dose-response studies revealed an ED(50) of 0 . 24-0.38 ng/ml with a maximal effect at 30 ng/ml . Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion . Biological effects of PTH-rp were evident in purified porcine theca cells . Using the Ca(2+)-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 microM) stimulated intracellular free calcium ion concentrations ({Ca(2+)}(i)) in single porcine theca cells . The {Ca(2+)}(i) elevation was characterized by a slow and prolonged rise . After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell {Ca(2+)}(i) response . Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-beta and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells . Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function . The precise role of this intraovarian PTH-rp system will require further study.

Free Radic Biol Med, 1999 Dec, 27(11-12), 1238 - 44
Effects of ischemia and H2O2 on the cold stress protein CIRP expression in rat neuronal cells; Xue JH et al.; Expression of CIRP (cold-inducible RNA-binding protein) is inducible at 32 degrees C in cultured fibroblasts . Because ischemia is known to induce expression of heat shock proteins, its effect on the CIRP expression was examined using the rat transient forebrain ischemia model . The isolated rat CIRP cDNA encoded amino acids 100% identical in its sequence to mouse CIRP . Northern blot analysis revealed that the CIRP transcripts were ubiquitously expressed in various tissues . In situ hybridization histochemistry of normal rat brain revealed the expression of CIRP in neurons in the hippocampus and the cerebral cortex among others . In the hippocampus of postischemic rats, CIRP mRNA level decreased from 3-6 h after the onset of reperfusion, while it did not change in the cerebral cortex . When PC12 pheochromocytoma cells were cultured at 32 degrees C, the CIRP mRNA level was increased . The presence of H2O2 in the culture media inhibited dose dependently this induction as well as constitutive expression, suggesting that the effect of brain ischemia on CIRP expression is related to generation of reactive oxygen species . Further studies are necessary to clarify the roles played by cold shock proteins in the hypothermic therapy of brain damages.

Br J Cancer, 2000 Jan, 82(1), 39 - 45
Androgen receptor protein is down-regulated by basic fibroblast growth factor in prostate cancer cells; Cronauer MV et al.; Interactions between polypeptide growth factors and the androgen receptor (AR) are important for regulation of cellular events in carcinoma of the prostate . Basic fibroblast growth factor (bFGF), the prototype of heparin-binding growth factors, and the AR are commonly expressed in prostate cancer . bFGF diminished prostate-specific antigen protein in the supernatants of androgen-stimulated human prostate cancer cells LNCaP by 80% . In the present study, we asked whether the bFGF effect on prostate-specific antigen is preceded by action on AR expression . LNCaP cells were treated with bFGF and AR protein expression was determined by immunoblotting and ligand binding assay . bFGF down-regulated AR protein in a dose-dependent manner showing a maximal effect at 50 ng ml(-1) both in the presence or absence of dihydrotestosterone . Down-regulation of AR protein expression occurred already after 8 h of bFGF treatment and a maximal inhibition was observed 24 h after addition of bFGF to culture media . As AR expression can be reduced by an increase in intracellular calcium levels, we investigated whether the bFGF effect on AR protein is mediated by this mechanism . Calcium release from intracellular stores and store-operated calcium influx after treatment with either bFGF or calcium ionophore A 23187 were measured by single cell fluorescence technique . The ionophore A 23187 was able to induce calcium influx and an increase in cytoplasmic calcium concentration in LNCaP cells . In contrast, bFGF was incapable of eliciting a similar effect . In contrast to AR protein, AR mRNA levels were not affected by bFGF as shown by semiquantitative reverse transcription polymerase chain reaction . In summary, these studies show that bFGF is a potent negative regulator of AR protein expression in the human prostate cancer cell line LNCaP.

Quintessence Int, 1999 May, 30(5), 351 - 6
In vitro volatile sulfur compound production of oral bacteria in different culture media; Quirynen M et al.; OBJECTIVE: The purpose of this study was to detect the relative contribution of Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia in the production of oral malodor . METHOD AND MATERIALS: The volatile sulfur compounds produced by these bacteria in vitro were measured semiquantitatively by a portable sulfide monitor . RESULTS: Samples from the tongue, tonsils, and pharynx showed a significantly higher production (550 ppb) of volatile sulfur compounds during the first 6 hours after anaerobic incubation in broths (brain-heart infusion, Columbia, and Trypticase Soy) than after incubation in agar media (300 ppb) (P < 0.001) . After 24 hours, values in broths and agars leveled off at 350 ppb (P = 0.3) and remained constant during the next 6 days . Measurement of separate pure cultures showed that maximal volatile sulfur compound production was reached 6 hours after incubation (450 ppb for the 3 bacteria) . Higher volatile sulfur compound values were measured in brain-heart infusion . When measurements of mixed cultures of the 3 pathogens were performed every 15 minutes, the maximal value was reached after only 30 minutes of incubation (nearly 500 ppb) . CONCLUSION: The in vitro volatile sulfur compound production of oral samples is preferably measured in broths . Maximal sulfur production from mixed cultures is reached after 30 minutes of incubation . Samples should always be inoculated at the same dilution.

J Mass Spectrom, 2000 Jan, 35(1), 13 - 22
Deuterium in vivo labelling of cytokinins in Arabidopsis thaliana analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry; Astot C et al.; A method was developed for analysing the biosynthetic rate of the cytokinin class of plant hormones . Transgenic, cytokinin-overproducing Arabidopsis thaliana plants were incubated in liquid culture media enriched with 30% deuterium oxide, and incorporation into the different parts of the cytokinin molecule was analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry after precolumn propionylation . The sugar moieties of the cytokinins generally showed a high and independent incorporation, so the analysis in this study focused on the cytokinin base moieties . It was observed that during a 24 h incubation period almost all labelling was incorporated into the side-chain, rather than the adenine moiety . The incorporation dynamics of isopentenyladenosine-5'-monophosphate, zeatinriboside-5'-monophosphate (ZRMP) and zeatin-9-glucoside were investigated through analysis of the cytokinin base fragments in high-resolution selective ion monitoring mode . Using a fractional synthetic rate approach, the biosynthetic rate of ZRMP was determined to be 18 ng h(-1) g(-1) fresh weight, giving a turnover time of 25 h . A method for the mass isotopomer abundance analysis of the cytokinins in the zeatin family, based on selective reaction monitoring, was also developed to gain further sensitivity . Use of this technique showed that there was a higher level of enrichment in zeatin nucleotide than in the corresponding nucleoside, in agreement with the hypothesis that cytokinin nucleotides are primary products in this pathway .

Int J Exp Pathol, 1999 Dec, 80(6), 349 - 55
Age-related changes in susceptibility of rat brain slice cultures including hippocampus to encephalomyocarditis virus; Su W et al.; Replication of the D variant of encephalomyocarditis virus (EMC-D) and its cytopathic effects were studied in the brain slice cultures including hippocampus (hippocampal slice) obtained from postnatal 1-, 4-, 7-, 14-, 28-and 56-day-old Fischer 344 rats . At 0, 12, 24, 36 and 48 h after infection, virus titres of the slices and culture media were assayed . Viral replication was observed in cultures from 1-to 28-day-old rats, and the highest titre was recorded in the slice and culture medium from the youngest rat . The peak of virus titre decreased with age and no distinct viral replication was observed in the cultures from 56-day-old rats . Light microscopy revealed that degenerative and necrotic changes appeared in the infected hippocampal slices from 1- to 28-day-old rats, and the changes became less prominent with age . In situ hybridization and indirect immunofluorescence staining showed that positive signals of viral RNA and antigen were prominent in younger rats and decreased with age . These results suggest that an age-related decrease in the susceptibility of rat brain to EMC-D is less related to the maturation of the immune system but possibly to that of the neurone.

Mol Cell Endocrinol, 1999 Dec 20, 158(1-2), 37 - 44
The effects of different culture media, glucose, pyridine nucleotides and adenosine on the activity of 11beta-hydroxysteroid dehydrogenase in rat Leydig cells; Ferguson SE et al.; 11Beta-hydroxysteroid dehydrogenase (11betaHSD) reversibly converts glucocorticoids into inert 11-ketosteroids . The direction of the reaction has been found to vary with the cell type and sub-cellular preparation used . We have investigated if the directionality of 11betaHSD can be influenced by the nature of the culture medium and compounds added during incubation of rat testis Leydig cells . We found that when the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) that the dehydrogenase (11betaDH) activity was higher than the reductase (11KSR) activity (11betaDH:11KSR ratio approximately 2:1) . When glucose was omitted from the DMEM a higher 11betaDH:11KSR ratio (approximately 33:1) was obtained . However, when the cells were cultured in a combination of DMEM/Ham's F12 (1:1, v/v), a ninefold increase in 11KSR activity was obtained whereas 11betaDH activity was inhibited by 64% compared with cells incubated in DMEM alone . Consequently, the predominant activity changed from a dehydrogenase to a reductase (11betaDH:11KSR ratio 1:15) . Addition of the individual components of the Ham's F12 medium to DMEM showed that only pyruvate and/or the amino acids were able to mimic the effects of DMEM/Ham's F12 . Similar differential effects were found when NAD+, NADH or adenosine were added to the Leydig cells incubated in DMEM (three to fivefold increases and 20-50% decreases in 11KSR and 11betaDH activities, respectively) . In contrast, NADP+ was found to increase 11betaDH activity (up to threefold) but NADPH had no effect on 11KSR activity . Cells incubated with DMEM/Ham's F12, NAD+, NADP+ and adenosine were found to have higher ATP levels (four to sixfold) than those incubated in DMEM alone . These results illustrate that the relative 11betaDH and 11KSR activities of 11betaHSD in Leydig cells are markedly and differentially altered by the nature of the incubation medium and compounds added.

J Lipid Res, 2000 Jan, 41(1), 12 - 22
Human crypt intestinal epithelial cells are capable of lipid production, apolipoprotein synthesis, and lipoprotein assembly; Levy E et al.; The recent availability of spontaneously proliferating, non-transformed human crypt intestinal epithelial cells (HIEC) affords an opportunity to investigate lipid metabolism in undifferentiated enterocytes . The major purpose of this study was to explore the capability of undifferentiated crypt cells to synthesize, assemble, and secrete lipids and apolipoproteins . HIEC were cultured in medium with 5% fetal bovine serum for 5 to 21 d . The cells were clearly able to incorporate {(14)C}oleic acid (dpm/mg protein) into triglycerides (128,279 +/- 16,988), phospholipids (30, 278 +/- 2,107), and cholesteryl esters (2,180 +/- 207) . Although improvement in lipid secretion was noted with prolongation of cell culture periods, low efficiency of lipid export (10.3 +/- 2.2% of intracellular content) characterized the HIEC . All phospholipid classes were elaborated, with phosphatidylcholine accounting for 79 . 3 +/- 1.3% of cellular phospholipids . Chylomicrons were the dominant (46.4%) lipoproteins secreted, followed by high, low, and very low density lipoproteins (HDL, LDL, and VLDL) comprising 22.5, 20.2, and 10.8% of the total, respectively . HIEC elaborated most of the major apolipoprotein (apo) classes (A-I, A-IV, B-100, C, and E), but were less efficient in producing apoB-48 . In contrast to the production of apoA-I and C as early as 5 days after confluence, apoA-I and A-IV were maximally expressed at 11 d . Culture media accumulated much more apoB-100 than apoB-48 (B-48/B-100 ratio 0.21 +/- 0.03), reflecting limited apoB mRNA editing . HIEC demonstrated both endogenous cholesterol synthesis and LDL receptor expression . Cholesterol synthesis was sensitive to 25-hydroxycholesterol and mevinolin, but unresponsive to LDL treatment, suggesting independent regulation pathways . In contrast, LDL inhibited receptor activity . The present findings provide the first solid evidence that immature HIEC are capable of key fat absorptive functions of well-differentiated enterocytes . The intracellular mechanisms required for lipid and apolipoprotein synthesis as well as for lipoprotein assembly are already present in intestinal crypt cells . These cells also retain the capacity for sterol enzyme and receptor expression . However, certain limitations, especially apoB-48 production and lipoprotein secretion as well as unresponsiveness of cholesterol synthesis to LDL, may be ascribed to the lack of differentiation.

Eye, 1999 Jun, 13 ( Pt 3a), 277 - 84
Re-establishment of visual circuitry after optic nerve regeneration; MacLaren RE; In mammals there are a few circumstances in which axotomised ganglion cell axons can regenerate . For instance, in vitro explants of retina can be encouraged to regenerate axons into appropriate culture media . Similarly, axotomised ganglion cells can regenerate into a peripheral nerve graft surgically connected to the optic nerve head, and during early development axons are able to regenerate across the retina to re-enter the optic nerve . This is certainly encouraging, but we are a long way from applying these observations to clinical practice . We need to know whether regenerating axons also retain a functional capacity for navigation . We must ask whether a regenerated projection is likely to be topographic rather than disordered . In this brief review we will look at some selected models of ganglion cell regeneration in order to examine this question of navigation in more detail . This is an important issue: the capacity to re-establish appropriate rather than random connections after ganglion cell regeneration would most likely be necessary for any meaningful return of visual function.

FEBS Lett, 1999 Dec 3, 462(3), 261 - 6
Expression, purification and characterization of recombinant mouse MT5-MMP protein products; Wang X et al.; We have recently identified the fifth member of the membrane-type matrix metalloproteinase subfamily, MT5-MMP/MMP24, which is expressed in a brain specific manner (Duanqing Pei (1999) J . Biol . Chem . 274, 8925-8932) . To further characterize its enzymic properties, an expression construct was engineered to produce MT5-MMP as a soluble and active form by truncating its transmembrane domain . Stable expression cell lines were subsequently established from MDCK cells transfected with this construct . Unfortunately, purification of MT5-MMP from the culture media in large quantity proves to be difficult initially due to its rapid turnover via a mechanism which can be inhibited by a broad spectrum metalloproteinase inhibitor, BB94 . Thus, BB94 was included in the cell culture medium and throughout the purification process except the final step of chromatography to protect MT5-MMP from destruction . Purified to homogeneity and free of the synthetic inhibitor, MT5-MMP can activate progelatinase A efficiently in a TIMP2 sensitive fashion . A preliminary screen for its potential substrates among extracellular matrix components identified the proteoglycans as the preferred substrates for MT5-MMP . Furthermore, it is determined that the stability of purified MT5-MMP is temperature dependent with rapid destruction at 37 degrees C, but being relatively stable at temperatures 4 degrees C or lower . These observations establish MT5-MMP as a proteoglycanase with a short half-life at body temperature, which may be critical for tightly controlled turnover of ECM components such as those in the brain.

Am J Clin Nutr, 2000 Jan, 71(1), 81 - 7
Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor alpha; Meerarani P et al.; BACKGROUND: Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent . OBJECTIVE: We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes . DESIGN: Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation . Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both . We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation . RESULTS: Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation . Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures . Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha . Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation . Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis . CONCLUSIONS: Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.

J Exp Zool, 2000 Feb 1, 286(2), 173 - 80
Developmental patterns of zygotes from transgenic female mice with elevated tissue glutathione; Rzucidlo SJ et al.; Zygotes were collected from transgenic mice overexpressing glutathione synthetase to test the hypothesis that such zygotes are more capable of developing in suboptimal culture media than zygotes from non-transgenic (control) mice . The effects of injection of donor mice with gamma-glutamylcysteinyl ethyl ester (gamma-GCE) on embryonic development were also investigated . In addition, the effects of genetic background (i.e., transgenic vs . non-transgenic) and injection with gamma-GCE on developmental capacity in kSOM were studied after exposure of zygotes to diamide (0.01 mM, for 30 min) . When cultured in modified Medium 16 significantly more pronuclear ova from transgenic females reached the morula (M) and early blastocyst (EB) stages than embryos derived from control mice . Genetic background significantly affected the proportions of embryos reaching 4- to 16-cell, M and EB stages during culture in modified Whitten's medium (WM); more zygotes collected from transgenic than from control mice developed . The injection of experimental mice with gamma-GCE significantly increased proportions of zygotes developing to M and EB stages in WM . Following exposure to diamide and subsequent culture in kSOM significantly more zygotes collected from transgenic mice reached the 4- to 16-cell stages than those from control females; a significant positive effect on developmental capacity was also seen after injection of donor mice with gamma-GCE . When cultured in suboptimal conditions, zygotes derived from transgenic mice overexpressing glutathione synthetase were more capable of developing than zygotes of non-transgenic control females . Zygotes from the transgenic mice also exhibited greater capacity to withstand toxic exposure to diamide . Present data suggest that commonly known strain differences in preimplantational development in vitro may reflect differences in the synthesis and/or metabolism of glutathione . J . Exp . Zool . 286:173-180, 2000 .

J Drug Target, 1999, 7(2), 143 - 56
Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes; Lucas P et al.; Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories . We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes . We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection . A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm) . Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media . The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm . This was thought to be due to aggregation, since neutral particles were most sensitive . When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells . Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different . Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive . This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore . Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate . Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium . Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine . This may explain the lower levels of transfection observed with high molecular weight polylysine . The significance of these observations for design of advanced non-viral gene delivery systems is discussed.

J Neurochem, 2000 Jan, 74(1), 114 - 24
Metabolic impairment elicits brain cell type-selective changes in oxidative stress and cell death in culture; Park LC et al.; Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases . Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response . Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells . The cells that have been implicated in TD-induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV-2 microglial cell lines, were cultured in either thiamine-depleted media or in normal culture media with amprolium, a thiamine transport inhibitor . The activity levels of a key mitochondrial enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons . The unique distribution of the activity did not account for the selective response to TD . TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells . Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation product . On the other hand, chronic lipopolysaccharide-induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells . The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.

Arthritis Rheum, 1999 Dec, 42(12), 2561 - 8
Fluoxetine and amitriptyline inhibit nitric oxide, prostaglandin E2, and hyaluronic acid production in human synovial cells and synovial tissue cultures; Yaron I et al.; OBJECTIVE: To evaluate the effects of fluoxetine and amitriptyline on nitric oxide (NO), prostaglandin E2 (PGE2), and hyaluronic acid (HA) production in human synovial cells and synovial tissue cultures . METHODS: Human synovial cells, synovial tissue, and cartilage were cultured in the presence or absence of cytokines, lipopolysaccharides (LPS), fluoxetine, or amitriptyline . Production of NO, PGE2, and HA was determined in culture media . Sulfated glycosaminoglycan (S-GAG) synthesis was evaluated in cartilage by 35S incorporation . RESULTS: Fluoxetine (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) inhibited NO release by 56%, 62%, and 71%, respectively, in the media of synovial cells stimulated by interleukin-1alpha (IL-1alpha; 1 ng/ml) plus tumor necrosis factor alpha (TNFalpha; 30 ng/ml) . Amitriptyline (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) caused a 16%, 27.3%, and 51.4% inhibition of NO release . Fluoxetine and amitriptyline (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) significantly (P<0.05) inhibited PGE2 release in the media of human synovial cells in the presence of IL-1alpha plus TNFalpha, in a dose-dependent manner (up to 88% inhibition) . Fluoxetine (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) and amitriptyline (1 microg/ml and 3 microg/ml) significantly (P<0.05) inhibited PGE2 release in the media of human synovial tissue in the presence of LPS . Fluoxetine and amitriptyline (0.3 microg/ml, 1 microg/ml, and 3 microg/ml) also significantly (P<0.05) inhibited HA production by human synovial cells in the presence of IL-1beta plus TNFalpha . Fluoxetine and amitriptyline (1 microg/ml) partially reversed IL-1beta-induced inhibition of 35S-GAG synthesis by human cartilage cultures (P<0.05) . Neither fluoxetine nor amitriptyline had a toxic effect on cells in the concentrations used . CONCLUSION: Inhibition of NO and PGE2 production by connective tissue cells is a mechanism by which some antidepressant medications may affect pain, articular inflammation, and joint damage.

Reprod Toxicol, 1999 Nov-Dec, 13(6), 547 - 54
Differential alteration by thalidomide of the glutathione content of rat vs . rabbit conceptuses in vitro; Hansen JM et al.; Thalidomide has been shown to cause limb reduction defects in rabbits with much greater potency than in rats, possibly due to inherent biochemical differences between the two species . Whole embryo culture was used to make direct comparisons between thalidomide-sensitive New Zealand White rabbits and thalidomide-resistant Sprague-Dawley rats, focusing on the possible roles of glutathione (GSH) and cysteine in mechanisms of thalidomide teratogenicity . Conceptuses were treated by adding thalidomide (0, 5, 15, and 30 microM) directly to the culture media containing conceptuses of similar gestational stages . Embryos and visceral yolk sacs (VYS) were measured for changes in GSH and cysteine content using HPLC after 24 h of exposure in vitro . Thalidomide-induced (15 and 30 microM) depletion of VYS GSH occurred only in the rabbit, where GSH concentrations (pmol/microg protein) fell significantly to about 50% of control . Rat VYS did not show a significant GSH depletion at any thalidomide concentration tested . Comparison between species showed that the control rabbit VYS contained 35% less GSH than the control rat VYS . Control rat embryos and control rabbit embryos contained similar concentrations of GSH, but thalidomide treatment preferentially depleted GSH in the rabbit at lower thalidomide concentrations (5 micro/M) . Cysteine concentrations were not significantly altered from control in the embryo or VYS of either species when treated with thalidomide . However, although control cysteine concentrations did not differ significantly between rat and rabbit VYS, control cysteine levels in rabbit embryos were 65% lower than those in control rat embryos . Rabbit conceptuses displayed lower species-specific GSH and cysteine levels and a greater propensity for thalidomide-induced GSH depletion than in rat conceptuses, consistent with the greater sensitivity of the rabbit to thalidomide teratogenicity . These thalidomide-induced and inherent species differences implicate a possible role for GSH and redox status in the mechanisms of thalidomide teratogenicity.

J Ocul Pharmacol Ther, 1999 Dec, 15(6), 525 - 35
Toxicity of pentoxifylline on monolayers of highly proliferative cells of epithelial origin; Ventura AC et al.; Interest in pentoxifylline has been recently reawakened owing to its suppressive effect on cell cytokine production . In this capacity, it may be of value as a routine supplement for culture media containing donor corneas . The purpose of the present study was to evaluate the toxic effects of pentoxifylline on two standardized cell lines of epithelial origin . Vero and Chang cells were incubated with various concentrations of pentoxifylline . Acute toxicity (4 hr) was assessed by monitoring the permeability of cells to propidium iodide; chronic toxicity (7 days) was determined by monitoring the effect of pentoxifylline on esterase activity and cell proliferation . The viability of cells was also assessed by microscopic inspection . Signs of acute toxicity became manifest at a pentoxifylline concentration of 100 mg/l in both Chang and Vero cells . Indications of chronic toxicity were observed at a drug concentration of 10 mg/l in Chang cells but at 1 mg/l in Vero ones . Proliferation was suppressed at pentoxifylline concentrations of 100 mg/l and 10 mg/l in Chang and Vero cells, respectively . Degenerative morphological changes were observed at a drug concentration of 100 mg/l in both cell types . At a concentration of 0.1 mg/l, pentoxifylline elicited no signs of acute or chronic toxicity in either Chang or Vero cells . At this dose, the drug is therefore unlikely to have deleterious effects on cultured donor corneas.

Mol Cell Endocrinol, 1999 Oct 25, 156(1-2), 45 - 53
Evidence for regulation of basic fibroblast growth factor gene expression by photoperiod and melatonin in the ovine pars tuberalis; Graham ES et al.; In the ovine pituitary the in vivo expression of the bFGF gene was studied using in situ hybridisation and Northern analysis . Expression of bFGF mRNA was highest in the pars tuberalis (PT) and zona tuberalis (ZT) and this expression was higher in the pituitaries of animals acclimited to long days than in those from short day housed animals . Expression in the pars intermedia (PI) was also detectable but the pars distalis (PD) showed negligible expression compared with the PT . Regulation of bFGF gene was investigated in primary cultured PT cells . Both forskolin and PMA increased bFGF mRNA expression significantly and melatonin was able to inhibit these effects partially, however there was no independent effect of melatonin on bFGF mRNA levels . Despite the inducibility of the bFGF gene, bFGF protein levels in PT culture media were insensitive to the same stimuli and detectable bFGF protein declined with time . Exogenous bFGF had no effect on c-fos mRNA levels and did not increase prolactin secretion from ovine lactotrophs . In contrast c-fos mRNA was induced in GH3 cells by bFGF . These data suggest that although basic fibroblast growth factor (bFGF) mRNA expression in the ovine PT is photoperiodically-sensitive, it is not directly involved in the seasonal regulation of lactotrophic activity.

Matrix Biol, 1999 Dec, 18(6), 523 - 32
The effect of mechanical strain on hyaluronan metabolism in embryonic fibrocartilage cells; Dowthwaite GP et al.; The development of the synovial joint cavity between the cartilage anlagen of the long bones is thought to be mediated by differential matrix synthesis at the developing articular surfaces . In addition, many studies have shown that removal of movement-induced mechanical stimuli from developing diarthrodial joints prevents cavity formation or produces a secondary fusion of previously cavitated joints . Herein, we describe an inductive influence of mechanical strain on hyaluronan metabolism and the expression of hyaluronan-binding proteins in cultured cells isolated from the articular surface of the distal tibial condyles of 18-day chick embryos . The effect of 10 min of mechanical strain on hyaluronan release into culture media, intracellular uridine diphospho-glucose dehydrogenase activity (an enzyme required for hyaluronan saccharide precursor production), cell surface hyaluronan-binding protein expression and HA synthase mRNA expression were analysed up to 24 h later . Six hours after the application of strain, there was a significant increase in the accumulation of hyaluronan released into tissue culture media by strained fibrocartilage cells compared with controls, an effect still detectable after 24 h . Strained cells also showed increased activity for uridine diphospho-glucose dehydrogenase and expressed higher levels of the hyaluronan-binding protein CD44 at 24 h . In addition, at 24 h mRNA for HA synthase 2 was expressed in all samples whereas mRNA for HA synthase 3 was only expressed in strained cells . These results further highlight the role for movement-induced stimuli in differential extracellular matrix metabolism during joint development and also show that strain may facilitate differential HA synthase gene expression.

Blood, 2000 Jan 1, 95(1), 173 - 9
Protein S secretion differences of missense mutants account for phenotypic heterogeneity; Espinosa-Parrilla Y et al.; To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated . Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis . Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S . Modestly low protein S was found in families with R520G and P626L (group B) mutants . T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants . The 6 protein S mutants were transiently expressed in COS 1 cells . The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%) . Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion . Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants . Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers . These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency . (Blood . 2000;95:173-179)

Oncol Rep, 2000 Jan-Feb, 7(1), 125 - 9
Alternative gonadotropin-releasing hormone processing products secreted from endometrial carcinoma; Takagi A et al.; Gonadotropin-releasing hormone (GnRH) receptor is demonstrated in uterine endometrial carcinoma . The endometrial carcinoma also produces GnRH or -like peptide, which prompted us to examine whether the intratumoral serves as natural ligand for its receptor . Endometrial carcinomas surgically removed had been screened for GnRH receptor expression before analysis . The in endometrial carcinoma cell-enriched culture media was characterized by immunoblots in tricine-supplied electrophoresis system and subsequent amino acid sequencing . Three major proteins of 10.0 kDa, 7.6 kDa and 1.1 kDa corresponding to pre-proGnRH, proGnRH and decapeptide GnRH, respectively, were detected in all of the ten endometrial carcinoma specimens tested . Immunoreactive contents in the culture media, assessed by RIA, ranged from 0.08 to 0.1 nM . In chorionic cell-conditioned media, only 1.1-kDa protein was detected . Endometrial carcinoma cells secrete alternative GnRH processing products in addition to natural GnRH . The GnRH variants may compete with mature GnRH at the level of its receptors, perhaps counteracting the GnRH signaling pathway to retard cell proliferation.

J Biol Chem, 1999 Dec 24, 274(52), 37270 - 9
Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast; Plant PJ et al.; Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (V-ATPase) is necessary for a variety of essential eukaryotic cellular functions . Nevertheless, yeasts lacking V-ATPase activity (Deltavma) are viable when grown at low pH, suggesting alternative methods of organellar acidification . This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging . When Deltavma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9 . Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis . Similarly, internalization of the plasmalemmal H(+)-ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the Deltavma phenotype and did not potentiate the vacuolar acidification . To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wild-type and the Deltavma yeasts were subjected to sudden changes in extracellular pH . In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5 . Importantly, these rapid pH changes were only observed when NH(4)(+), routinely added as a nitrogen source, was present . The NH(4)(+)-dependent acidification was not due to efflux of NH(3) from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH(4)(+) . These findings suggest that although NH(3) can act as a cell-permeant proton scavenger, NH(4)(+) may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast . The high concentration of NH(4)(+) frequently added as a nitrogen source to yeast culture media together with effective NH(4)(+) transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.

Hum Reprod, 1999 Dec, 14(12), 3069 - 76
Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro; Sjoblom C et al.; The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized in the female reproductive tract and has been implicated in the growth and development of the preimplantation embryo in rodent and livestock species . To examine the effect of GM-CSF on human embryo development in vitro, surplus frozen 2-4-cell embryos were cultured in media supplemented with 2 ng/ml recombinant human GM-CSF . The addition of cytokine increased the proportion of embryos that developed to the blastocyst stage from 30 to 76% . The developmental competence of these blastocysts, as assessed by hatching and attachment to extracellular matrix-coated culture dishes, was also improved by GM-CSF . The period in culture required for 50% of the total number of blastocysts to form was reduced by 14 h, and blastocysts grown in GM-CSF were found to contain approximately 35% more cells, due primarily to an increase in the size of the inner cell mass . The beneficial effect of GM-CSF was exerted in each of two sequential media systems (IVF-50/S2 and G1 . 2/G2.2) and was independent of the formulation of recombinant cytokine that was used . These data indicate that GM-CSF may have a physiological role in promoting the development of the human embryo as it traverses the reproductive tract in vivo, and suggest that addition of this cytokine to embryo culture media may improve the yield of implantation-competent blastocysts in human in-vitro fertilization programmes.

Am J Physiol, 1999 Dec, 277(6 Pt 2), F934 - 47
Role of membrane-type matrix metalloproteinase 1 (MT-1-MMP), MMP-2, and its inhibitor in nephrogenesis; Kanwar YS et al.; Extracellular matrix (ECM) proteins, their integrin receptors, and matrix metalloproteinases (MMPs), the ECM-degrading enzymes, are believed to be involved in various biological processes, including embryogenesis . In the present study, we investigated the role of membrane type MMP, MT-1-MMP, an activator pro-MMP-2, in metanephric development . Also, its relationship with MMP-2 and its inhibitor, TIMP-2, was studied . Since mRNAs of MT-1-MMP and MMP-2 are respectively expressed in the ureteric bud epithelia and mesenchyme, they are ideally suited for juxtacrine/paracrine interactions during renal development . Northern blot analyses revealed a single approximately 4.5-kb mRNA transcript of MT-1-MMP, and its expression was developmentally regulated . Inclusion of MT-1-MMP antisense oligodeoxynucleotide (ODN) in the culture media induced dysmorphogenetic changes in the embryonic metanephros . MMP-2 antisense ODN also induced similar changes, but they were relatively less; on the other hand TIMP-2 antisense ODN induced a mild increase in the size of explants . Concomitant exposure of MT-1-MMP and MMP-2 antisense ODNs induced profound alterations in the metanephroi . Treatment of TIMP-2 antisense ODN to metanephroi exposed to MT-1-MMP/MMP-2 antisense notably restored the morphology of the explants . Specificity of the MT-1-MMP antisense ODN was reflected in the selective decrease in its mRNA and protein expression . The MT-1-MMP antisense ODN also resulted in a failure in the activation of pro-MMP-2 to MMP-2 . These findings suggest that the trimacromolecular complex of MT-1-MMP:MMP-2:TIMP-2 modulates the organogenesis of the metanephros, conceivably by mediating paracrine/juxtacrine epithelial:mesenchymal interactions.

Thromb Haemost, 1999 Nov, 82(5), 1497 - 503
Natriuretic peptides regulate the expression of tissue factor and PAI-1 in endothelial cells; Yoshizumi M et al.; In the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II . The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay . Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1 . However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.

J Comp Physiol {B}, 1999 Oct, 169(7), 515 - 20
Differences in fuel utilization between trout and human thrombocytes in physiological media; Guppy M et al.; Cell culture preparations now play a significant and essential role in physiological and biochemical studies of cell biology . However, the fuels offered in cell culture media are only glucose and glutamine, plus whatever might be in the added sera . It is currently difficult to find a rational way forward on this problem, as there are few data on what fuels cells use in vivo or even in an in vitro physiological situation . A recent study on human platelets redressed the situation somewhat by finding that 75% of ATP turnover could be accounted for by aerobic glycolysis, and by the oxidation of glucose, hydroxybutyrate, acetate, glutamine, palmitate and oleate . In the present study we used a similar strategy to investigate fuel choices by trout thymocytes, cells with a similar function but from a different phylogenetic group . When these cells were presented with a physiological medium, we found that aerobic glycolysis accounted for 9% of total ATP turnover, glucose and glutamine oxidation made a combined contribution of 2.3%, oleate and palmitate oxidation accounted for 15%, and 74% was unaccounted for . These patterns of fuel use are very different from that in human platelets . They demonstrate the cell- and animal-specific nature of cellular metabolism and again expose the inadequacy of the fuel component in culture media.

J Cell Sci, 2000 Jan, 113 ( Pt 1), 113 - 21
Source, catabolism and role of the tetrapeptide N-acetyl-ser-asp-lys-Pro within the testis; Stephan J et al.; The tetrapeptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP) is a natural regulator of hematopoietic stem cell proliferation . The present study was aimed at investigating the presence and the role of AcSDKP in rat testis . Specific immunoreactivity was always observed in the interstitial tissue at all stages of testicular development and in elongated spermatids at 45 days of age and in adults . In accordance with the interstitial labeling, high AcSDKP levels were detected in Leydig cell and testicular macrophage culture media and cell extracts, as well as in the testicular interstitial fluid (TIF) . Much lower concentrations were found in peritubular cells and Sertoli cells cultures, whereas very low concentrations were present in cultured spermatocytes and spermatids . In contrast to the slight degradation rate of AcSDKP observed in the spermatocyte and spermatid culture media, no catabolism of the peptide was seen in testicular somatic cell culture medium . Furthermore, the degradation rate of AcSDKP was much lower in TIF than in peripheral blood plasma . Despite the very strong evidence indicating that Leydig cells and testicular macrophages produce AcSDKP, the selective destruction of these cells did not result in any change in AcSDKP levels in TIF or in plasma . This suggests a compensatory mechanism ensuring constant levels of the peptide in TIF when interstitial cells are absent . Finally, in vitro, in the presence of AcSDKP, significantly more {(3)H}thymidine incorporation was found in A spermatogonia . In conclusion, this study establishes the presence of very high concentrations of AcSDKP in rat testis and demonstrates its Leydig cell and testicular macrophage origin . The presence of AcSDKP in the TIF and its stimulatory effect on thymidine incorporation in spermatogonia very strongly suggest its implication in the paracrine control of spermatogenesis.

Int J Dev Neurosci, 1999 Dec, 17(8), 787 - 93
Role of acetylcholinesterase in the development of axon tracts within the embryonic vertebrate brain; Anderson RB et al.; In the developing vertebrate brain, acetylcholinesterase (AChE) expression coincides temporally with axon tract formation . Although AChE promotes neurite outgrowth in vitro, the role of this molecule in the development of axon tracts in vivo is unknown . To address this question, we examined the effects of the AChE inhibitor, BW284C51, on the formation of the early scaffold of axon tracts in the embryonic Xenopus brain . In exposed Xenopus brain preparations, axons elongate and establish a normal topography of axon tracts . However, when brains were exposed to BW284C51, the thickness of the major longitudinal axon tract, the tract of the post-optic commissure decreased in a dose-dependent manner . When BW284C51 was removed from the culture media axon tract development returned to normal within 5 h . These findings provide the first evidence for a non-classical role of AChE in the initial formation of axon tracts within the developing vertebrate brain.

Biochem J, 1999 Dec 15, 344 Pt 3, 643 - 8
The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack; Le Goffe C et al.; This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack . Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e . anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power . Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate {3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine} only in galactose-containing medium . In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation . The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl . 16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by (14)CO(2) production from 2-deoxy{1-(14)C}glucose . Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury . Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.

Blood, 1999 Dec 15, 94(12), 4122 - 31
Hypofibrinogenemia associated with a heterozygous missense mutation gamma153Cys to arg (Matsumoto IV): in vitro expression demonstrates defective secretion of the variant fibrinogen; Terasawa F et al.; We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency . Direct sequencing of a polymerase chain reaction-amplified gamma-chain gene segment showed a novel nucleotide substitution . This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153 . To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant gamma-chain DNAs encoding gamma153R and gamma153A for in vitro expression in Chinese hamster ovary (CHO) cells . Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with gamma153R or gamma153A synthesized the variant gamma-chain, but did not secrete variant fibrinogen into the culture medium . Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant gamma-chain was expressed . In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with (35)S-methionine . Neither intermediates nor intact fibrinogen was seen with the variant gamma-chains . These data suggest that gamma-chains have an important early role in fibrinogen assembly . Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of alphagamma or betagamma dimers, or both . This model implies that gammaCys153 has a critical role in the formation of these early assembly intermediates . We concluded that the gamma153Cys-->Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency . We designated this variant as fibrinogen Matsumoto IV.

Immunology, 1999 Nov, 98(3), 363 - 70
Nitric oxide and macrophage antiviral extrinsic activity; Benencia F et al.; In this study we evaluated the relationship between nitric oxide (NO) and macrophage antiviral extrinsic activity . Macrophages activated by intraperitoneal injection of herpes simplex virus-2 (HSV-2), showed both extrinsic antiviral activity and high nitrite production in contrast to non-activated, resident macrophages . The extrinsic antiviral activity was observed in cultures of Vero cells infected with HSV-1 and HSV-2 . The NO inhibitor N-monomethyl-l-arginine acetate (l-NMA) impaired the antiviral activity of HSV-elicited macrophages . The effect was dose dependent and correlated with a reduction of nitrite in the culture media . The effect of l-NMA was reversed by the addition of l-arginine . These data indicate that NO could be responsible for the described activity . Furthermore, l-NMA treatment resulted in the aggravation of HSV-1-induced keratitis in the mouse model, supporting a defensive role of NO in the pathogenesis of HSV-1 corneal infection.

Scand J Gastroenterol, 1999 Nov, 34(11), 1132 - 8
Peripheral blood mononuclear cells promote intestinal epithelial restitution in vitro through an interleukin-2/interferon-gamma-dependent pathway; Cario E et al.; BACKGROUND: Acute intestinal mucosal inflammation is associated with recruitment of peripheral blood mononuclear cells (PBMN) into the mucosa and migration across the epithelium . It has recently been shown that several PBMN-derived cytokines, including transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) may modify intestinal epithelial cell function, resulting in rapid improvement of wound repair . Our aim was to characterize the modulating effects of PBMN on intestinal epithelial restitution, the initial step of wound healing . METHODS: PBMN were separated from whole blood, obtained from healthy volunteers, by using a density gradient . The effect of PBMN on intestinal epithelial restitution was assessed by using an in vitro coculture wounding model with non-transformed small-intestinal epithelial IEC-6 cells . RESULTS: Coculture of PBMN caused a significant enhancement of epithelial cell restitution in vitro . The modulatory effects of PBMN could be significantly blocked by adding immunoneutralizing anti-IL-2 or anti-IFN-gamma to the culture media, suggesting that PBMN may modulate intestinal epithelial migration through an IL-2- and IFN-gamma-dependent pathway . In contrast, PBMN-induced stimulation of intestinal epithelial restitution was not influenced by addition of anti-TGF-beta or anti-TNF-alpha, suggesting that these cytokines are not critical for the modulation of restitution by PBMN . CONCLUSIONS: These findings suggest that PBMN may promote intestinal epithelial wound repair by enhancing restitution through secretion of various cytokines, among them IL-2 and IFN-gamma, which are abundantly expressed in the course of several inflammatory diseases of the gut.

Scand J Gastroenterol, 1999 Nov, 34(11), 1099 - 102
31-43 amino acid sequence of the alpha-gliadin induces anti-endomysial antibody production during in vitro challenge; Picarelli A et al.; BACKGROUND: Wheat gliadin is the culprit antigen of coeliac disease (CD) . Two short sequences of NH2-terminal portion of gliadin seem to be responsible for CD . Antiendomysial antibodies (EMA), highly sensitive and specific for CD, are detectable in the culture media from treated CD patients, after in vitro challenge with peptic-tryptic (PT) digest of gliadin . In this study we detected EMA production after in vitro challenge with 31-43 peptide . We used 56-68 peptide, lacking toxic sequences, as a negative control . METHODS: Duodenal samples from 11 treated CD patients and 9 control patients were cultured with 31-43 and 56-68 peptides and PT gliadin . Indirect immunofluorescence analysis was used for EMA detection . RESULTS: EMA were detected in culture media of 10 of 11 specimens challenged with PT-gliadin and in the media of all specimens challenged with 31-43 peptide . No EMA were detectable in any treated patients cultured with 56-68 peptide or with medium alone . No EMA were observed in cultures of control specimens . DISCUSSION: The ability of the 31-43 sequence of the alpha-gliadin to induce EMA production suggests its involvement in the pathogenesis of CD . Furthermore, it may be a more useful antigenic substance than PT gliadin for both in vitro and in vivo studies of CD.

Rev Med Brux, 1999 Oct, 20(5), A463 - 7
{Diminishing the risk of multiple pregnancies in in vitro fertilization: from selective transfer of two embryos to that of one blastocyst?}; Devreker F et al.; The risk of multiple pregnancy after IVF needs to be drastically reduced . Several policies can be applied including the transfer of a maximum of three embryos to all patients, the fertilization of a maximum of three oocytes or a selective reduction of the number of transferred embryos . The first policy previously applied at the Fertility Clinic at Erasme Hospital until 1996, transferred two good quality embryos to patients with at least three good embryos . If this policy demonstrated that patients with two transferred embryos had similar chances of pregnancies compared to patients with three transferred embryos, it failed to sufficiently decrease the number of multiple pregnancies . The second policy applied since 1997, transferring a maximum of two average or good embryos to all patients aged under 35 years and with less than 3 previous attempts, demonstrated that while preserving the chances of pregnancy for these patients, it decreased by 20% the number of multiple pregnancies and almost eliminated triplets . With the improvement of culture media, it is now possible to culture embryos in vitro for a longer period and therefore transfer embryos with proven viability at a time corresponding more to in vivo physiological conditions . The implantation rates for these embryos, for patients with at least 4 previous attempts can reach 40% . If these results persist, it would be possible to transfer blastocysts to all patients and perhaps move on to the replacement of a single embryo, a policy that will practically eradicate all multiple pregnancies.

Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 268 - 73
Hyaluronidase expression in human skin fibroblasts; Stair-Nawy S et al.; Hyaluronidase activity has been detected for the first time in normal human dermal fibroblasts (HS27), as well as in fetal fibroblasts (FF24) and fibrosarcoma cells (HT1080) . Enzymatic activity was secreted predominantly into the culture media, with minor amounts of activity associated with the cell layer . In both classes of fibroblasts, hyaluronidase expression was confluence-dependent, with highest levels of activity occurring in quiescent, post-confluent cells . However, in the fibrosarcoma cell cultures, expression was independent of cell density . The enzyme had a pH optimum of 3.7 and on hyaluronan substrate gel zymography, activity occurred as a single band corresponding to an approximate molecular size of 57 kDa . The enzyme could be immunoprecipitated in its entirety using monoclonal antibodies raised against Hyal-1, human plasma hyaluronidase . PCR confirmed that fibroblast hyaluronidase was identical to Hyal-1 . The conclusion by previous investigators using earlier technologies that fibroblasts do not contain hyaluronidase activity should be reevaluated .

J Neuroimmunol, 1999 Nov 15, 101(2), 148 - 60
Increased expression of bioactive chemokines in human cerebromicrovascular endothelial cells and astrocytes subjected to simulated ischemia in vitro; Zhang W et al.; Leukocyte infiltration into the brain has been implicated in the development of ischemic brain damage . In this study, simulated in vitro ischemia/reperfusion and IL-1beta were found to up-regulate both the expression of intercellular adhesion molecule- (ICAM-1) in cultured human cerebromicrovascular endothelial cells (HCEC) and the adhesion of allogenic neutrophils to HCEC . Both HCEC and human fetal astrocytes (FHAS) also responded to IL-1beta and to in vitro ischemia/reperfusion by a pronounced up-regulation of IL-8 and MCP-1 mRNA and by increased release of IL-8 and MCP-1 in cell culture media . FHAS were found to release 30-times higher levels of MCP-1 than HCEC under both basal and ischemic conditions . However, 100 u/ml IL-1beta induced greater stimulation of both IL-8 and MCP-1 secretion in HCEC (50 and 20 times above controls, respectively) than in FHAS (three and two times above controls, respectively) . IL-8 was the principal neutrophil chemoattractant released from IL-1beta-treated HCEC, since IL-8 antibody completely inhibited neutrophil chemotaxis enticed by HCEC media . However, the IL-8 antibody neutralized only 50% of IL-1beta-stimulated neutrophil chemoattractants released from FHAS, and 40%-60% of ischemia-stimulated chemotactic activity released by either HCEC or FHAS . These results suggest that simulated in vitro ischemia, in addition to IL-8 and MCP-1, stimulates secretion of other bioactive chemokines from HCEC and FHAS.

Lipids, 1999 Oct, 34(10), 1031 - 6
Solubilization and stabilization of carotenoids using micelles: delivery of lycopene to cells in culture; Xu X et al.; The use of the organic cosolvents tetrahydrofuran and dimethylsulfoxide was found to be unsuitable for prostate tumor cell cultures because of solvent cytotoxicity and the poor solubility and instability of lycopene . For example, the half-life of lycopene in organic/aqueous solution was found to be less than 2 h . Therefore, a micellar preparation of lycopene was developed for the solubilization and stabilization of lycopene in cell culture media . Neither the micelles themselves nor lycopene solubilized in micelles at concentrations up to 10 microg/mL in the cell culture media produced cytotoxicity or inhibition of cell proliferation in either LNCaP human prostate cells or Hs888Lu human lung cells . Lycopene solubilized in micelles was stable for at least 96 h under standard cell culture conditions so that a constant lycopene supply could be provided to the cells . During the culture process, lycopene was taken up by LNCaP cells and reached a plateau at approximately 12 h . Micelles provide a convenient, inexpensive, and nontoxic vehicle for dissolving and stabilizing carotenes such as lycopene in tissue culture media and then delivering them to cells growing in culture.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1999 Sep, 124(1), 109 - 16
Hormonal profiles correlated with season, cold, and starvation in Rana catesbeiana (bullfrog) tadpoles; Wright ML et al.; Bullfrog (Rana catesbeiana) tadpoles are of value to amphibian researchers because of their large size, and year-round availability due to overwintering in many latitudes . Concern over a possible difference in hormonal parameters in tadpoles obtained at different times of the year prompted us to investigate thyroid gland secretion in vitro, plasma and ocular melatonin, and plasma corticosteroids in late pre- to early prometamorphic larvae on 12L:12D . Winter tadpoles exposed to 22 degrees C for 3 weeks of acclimation (winter group) were compared to summer tadpoles kept at 22 degrees C (summer group), as well as to summer tadpoles exposed to cold (12 degrees C) for the 3 weeks (cold group), or kept at 22 degrees C and starved for the last week of acclimation (starved group) . Thyroids from the summer group had a significantly higher response to 0.2 microg/ml ovine thyrotropin (TSH) than the other groups, indicating that cold and starvation inhibited subsequent in vitro thyroid sensitivity to TSH . The thyroids of the starved tadpoles had significantly higher baseline (unstimulated) thyroxine (T4) secretion into the culture media, a finding that might be related to starvation-induced acceleration of metamorphosis . Plasma melatonin was lower, and ocular melatonin significantly higher in both summer and starved groups, while the reverse occurred in the winter and cold groups . Thus, seasonal or induced cold brought on a shift in the relationship of plasma to ocular melatonin but starvation had no effect . There were no significant differences among the treatment groups in plasma hydrocortisone (HC) and aldosterone (ALDO) levels, except that HC was lower than ALDO only in the plasma of winter tadpoles . We conclude that seasonal variation needs to be taken into account in endocrine experiments utilizing tadpoles obtained at different times of the year.

Scand J Infect Dis, 1999, 31(5), 485 - 7
Improved recovery of Mycobacterium tuberculosis from pleural aspirates: bedside inoculation, heparinized containers and liquid culture media; Cheng AF et al.; The effects of bedside inoculation, heparinized containers and liquid culture media on the recovery of Mycobacterium tuberculosis from pleural aspirates were evaluated in this study . Of 155 patients, 63 were diagnosed to have pleural effusion tuberculous in origin . The overall recovery of M . tuberculosis was 57.1% . Bedside inoculation of the specimens produced a significantly higher yield than laboratory inoculation using non-heparinized specimens . When the pleural aspirates were transported in heparinized containers, the recovery rate was comparable to that from bedside inoculation, but lower when non-heparinized containers were used . No significant difference was found in recovery rate between the two liquid media, but the rate was significantly higher with the use of liquid media than conventional solid media . Thus, bedside inoculation of pleural aspirates, use of heparinized containers for transport for delayed inoculation in the laboratory and use of liquid culture media are recommended.

J Bone Miner Metab, 1999, 17(4), 245 - 51
Tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) stimulate osteoclastic bone resorption; Shibutani T et al.; As both tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 have been reported to inhibit bone resorption, we examined whether TIMP-1 or TIMP-2 in fetal calf serum (FCS), with which culture media were supplemented, affected osteoclastic bone resorption in vitro . Contrary to our expectation, almost complete suppression of osteoclastic bone resorption was observed when both TIMP-1 and TIMP-2 were removed from the FCS . Bone resorption was, however, almost fully restored by the addition of recombinant TIMPs . TIMPs stimulate bone resorption at significantly lower concentrations (approximately ng/ml) than those (approximately micrograms/ml) required to inhibit bone resorption . To understand the mechanism of TIMP-dependent bone resorption, we counted and compared the number of tartrate-resistant acid phosphatase-(TRAP-) positive and multinuclear cells in cultures containing either 10% FCS or TIMP-1-free and/or TIMP-2-free FCS . There was essentially no difference in number among these, suggesting that the TIMP role seems to be related to the functional expression of osteoclasts . Metalloproteinase inhibitors, either BE16627B{L-N-(N-hydroxy-2-isobutylsuccinynamoyl)-seryl-L-val ine} or R94138 inverted question markN-methyl-(3S)-2-{(2R)-2-hydroxycarbamoylmethylundecanoyl} hexahydropyridazine-3-carboxamide inverted question mark, could not replace TIMPs, suggesting that the osteoclast-stimulating activity of TIMPs cannot be ascribed to merely their inhibitory effect on matrix metalloproteinases.

J Assist Reprod Genet, 1999 Nov, 16(10), 529 - 34
Clinical outcome of day 2 versus day 3 embryo transfer using serum-free culture media: a prospective randomized study; Ertzeid G et al.; PURPOSE: The objective was to evaluate whether extending the embryo culture period from 2 to 3 days would yield a more optimal selection of viable embryos, thereby increasing the implantation and live birth rates . METHODS: Patients undergoing in vitro fertilization with at least one oocyte fertilized were prospectively randomized to 2 or 3 days of embryo culture in serum-free media . On the basis of their morphology and cleavage rate, a maximum of three embryos was selected for transfer . RESULTS: Embryos transferred on day 2 or day 3 were similar morphologically, however, a higher proportion of retarded embryos was observed on day 3 . The implantation rate was 15.8 and 14.3% for day 2 and day 3 transfers, respectively . The increase in live birth rate from 18.5 to 22.6%, possibly suggesting a better embryo selection on day 3, was not statistically significant . CONCLUSIONS: Extending the embryo culture period from 2 to 3 days had no effect on implantation and live birth rates.

J Gastroenterol Hepatol, 1999 Nov, 14(11), 1062 - 9
Ethanol intake preceding Helicobacter pylori inoculation promotes gastric mucosal inflammation in Mongolian gerbils; Suzuki H et al.; BACKGROUND: Mongolian gerbils have been reported to be a suitable model for Helicobacter pylori-associated gastric mucosal injury, including gastric cancer . Although ethanol is known to be one of the harmful substances in the gastric mucosa, the relationship between ethanol and H . pylori infection remains unknown . The aim of the present study is to investigate the effect of ethanol treatment prior to H . pylori inoculation on associated gastric mucosal injury . METHODS: Male Mongolian gerbils were used for the study . Helicobacter pylori was orally inoculated after 15 h fasting (Hp group) . Thirty minutes prior to H . pylori inoculation, a group of gerbils was orally treated with 40% ethanol (20 mL/kg; E + Hp group) . Another group of animals was treated either with H . pylori culture media alone (controls) or with 40% ethanol plus culture media (E group) . Gerbils were killed 2, 4 or 12 weeks after H . pylori inoculation . Helicobacter pylori infection was confirmed by both histological examination and serological tests . Mucosal damage was evaluated histologically according to the modified Sydney system . RESULTS: Although in the controls and E group no significant change to the gastric mucose was observed, persistent H . pylori infection was seen in the mucosa and mucosal leucocyte infiltration and severe epithelial damage was observed in the Hp and E + Hp groups after 4 weeks . The histological scores for polymorphonuclear cell infiltration and myeloperoxidase activity were higher in the E + Hp group at 4 weeks than in the Hp group (P < 0.05) . CONCLUSIONS: Ethanol intake preceding H . pylori inoculation could promote the progression of gastric mucosal inflammation in Mongolian gerbils.

Hum Reprod, 1999 Sep, 14 Suppl 1, 145 - 61
Oocyte in-vitro maturation and follicle culture: current clinical achievements and future directions; Smitz J et al.; This paper reviews two recent developments in the field of assisted reproduction: in-vitro culture (IVC) of follicles and in-vitro maturation (IVM) of oocytes . Both in-vitro procedures for oocytes and immature follicles were developed so as to enable storage of ovarian cortical tissue and oocyte-cumulus complexes (OCC) from small follicles . Until now, complete in-vitro development from a primordial follicle up to ovulation has been achieved only in mice . Culture of ovarian cortex from large mammals and humans has revealed that the proliferation and differentiation of pre-granulosa cells is easily switched on, but that within a few days oocyte growth is often compromised . Culture of OCC from preantral follicles from sheep yielded antral follicles after 1 month in a serum-free system and the enclosed oocytes had almost doubled their diameter . Similar work in humans is ongoing, but no consistent successes have been reported as yet . In-vitro maturation of OCC from antral follicles has already been used clinically for more than 10 years . Inspired by the work on bovines, several in-vitro fertilization (IVF) clinics have applied IVM on OCC from different sizes of follicles . The culture conditions needed to provide a reasonable yield of developmentally competent oocytes have not been clearly defined, although some IVF groups have reported live births . To enable the development of consistent techniques of IVC and IVM, a more systematic and scientific approach is needed: use of defined culture media in combination with follicle stage-dependent supplements might improve our understanding of the minimal requirements for oocyte growth and maturation.

Am J Respir Cell Mol Biol, 1999 Dec, 21(6), 666 - 74
Bronchial tissue kallikrein activity is regulated by hyaluronic acid binding; Forteza R et al.; Tissue kallikrein (TK) is secreted by serous cells of tracheobronchial submucosal glands and plays a role in allergic airway responses . To better understand the regulation of TK, we used primary cultures of submucosal gland cells that release TK upon stimulation . Media from cultures stimulated with chymase (10(-7) M) showed increased TK activity (0.50 +/- 0.22 mU/ml mean +/- standard error) in comparison with the control group (0.08 +/- 0.02 mU/ml) . The increased TK activity was significantly correlated with increases in the levels of the serous cell marker, secretory leukoprotease inhibitor . Anion exchange chromatography of the conditioned culture media showed that TK activity eluted as a broad peak between 1.6 and 1.8 M NaCl, unlike the reported elution (0.3 to 0.6 M NaCl) of kallikreins from other tissues, suggesting that secreted bronchial TK was bound to a negatively charged molecule . Hyaluronidase digestion increased TK activity in both pre- and post-chymase-stimulated culture media, whereas no such change was seen after samples were digested with heparinase or chondroitinase ABC . Further, after hyaluronidase digestion of media, TK eluted from an anion exchange column between 0.3 and 0.6 M NaCl . Enzymatic detection of TK after nondenaturing gel electrophoresis showed that hyaluronidase digestion also reduced the electrophoretic heterogeneity of TK to a single band, whereas adding back hyaluronic acid (HA) to hyaluronidase-digested samples restored the original heterogeneity . Finally, TK activity bound to HA-Sepharose and could be eluted with HA . These studies show that primary cultures of ovine submucosal gland cells secrete TK in a regulated fashion, and that secreted TK binds to HA . This binding reduces TK enzymatic activity; therefore, factors that affect HA turnover could modify the TK activity in the airway lumen . These events could be important in the regulation of kinin-mediated airway inflammation.

FEBS Lett, 1999 Oct 22, 460(1), 23 - 6
Transforming growth factor-beta1 inhibits expression of selenoprotein P in cultured human liver cells; Mostert V et al.; The effect of cytokines on the expression of selenoprotein P (SeP) in the human liver cell line HepG2 was investigated . Treatment with interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha had no effect on SeP levels in culture media or on SeP mRNA expression . Conversely, Western analysis revealed a dose-dependent reduction of SeP content in culture medium after treatment with transforming growth factor (TGF)-beta1 with an 1C50 of 31 pM . Treatment with 100 pM TGF-51 for 48 h led to a decrease to 21 +/- 9% of controls . RT-PCR analysis of SeP mRNA expression demonstrated an inhibition of SeP transcription to 40+/-2% of control levels after 24 h . The expression of a luciferase reporter construct under control of the human SeP promoter was downregulated by TGF-beta1 treatment in a dose-dependent fashion indicating a transcriptional regulation of the SeP gene by TGF-beta1.

J Pharmacol Exp Ther, 1999 Dec, 291(3), 1276 - 83
4-methylcatechol increases brain-derived neurotrophic factor content and mRNA expression in cultured brain cells and in rat brain in vivo; Nitta A et al.; Practical use of brain-derived neurotrophic factor (BDNF) as therapy is limited by two serious problems, i.e., its inability to cross the blood-brain barrier and its instability in the bloodstream . In the present study, we investigated the effects of 4-methylcatechol (4-MC), which stimulates nerve growth factor synthesis and protects against peripheral neuropathies in rats, on BDNF content and mRNA expression in cultured brain cells and in vivo in the rat brain . 4-MC elevated BDNF content in culture media of both rat astrocytes and neurons with different dose-response relations . The increase in BDNF mRNA level was correlated with the increase in BDNF content, demonstrating that 4-MC can stimulate BDNF synthesis of both neurons and astrocytes . Then we examined the in vivo effects of 4-MC . First, we found that ventricularly administered 4-MC facilitated an increase in the BDNF content in the cerebral cortex and hippocampus in association with its diffusion into the brain parenchyma . Second, i.p . administration of 4-MC enhanced BDNF mRNA expression in the infant rat brain, in which the blood-brain has not yet fully been established . These results demonstrate that 4-MC, once delivered into the brain, can stimulate BDNF synthesis.

Gen Comp Endocrinol, 1999 Nov, 116(2), 249 - 60
Influence of cortisol on the larval bullfrog thyroid axis in vitro and in vivo and on plasma and ocular melatonin; Wright ML et al.; Adrenal (interrenal) steroids have an important role in amphibian development, antagonizing the metamorphic changes induced by the thyroid at first and then synergizing with the thyroid hormones as their level rises during metamorphosis . Because most of the studies of corticoids at metamorphosis have focused on peripheral tissues, we investigated the effect of cortisol (hydrocortisone; HC) in vitro and in vivo on the thyroid of Rana catesbeiana (bullfrog) tadpoles on 12:12 light/dark (LD) cycles . Plasma and ocular melatonin, which is altered by changes in thyroxine (T(4)) levels, were also assayed in some experiments . Thyroids from premetamorphic tadpoles secreted less T(4) into culture media when incubated with 10 micrograms/ml HC and 0.2 micrograms/ml ovine thyrotropin (TSH) than with TSH alone and when cultured in the absence of TSH following 5 days of 10-micrograms HC injections, indicating that HC inhibited the thyroid at young stages . The effect of 10 micrograms/ml HC at older stages was investigated by culturing thyroids and pituitaries separately on the first day in control or HC media and then incubating the thyroids on the second day in homologous pituitary-conditioned media as a bioassay for pituitary TSH . HC had no effect on baseline T(4) secretion by the thyroids of prometamorphic or climax tadpoles on the first day but increased T(4) secretion over the control on the second day . Thyroids cultured with TSH and HC showed no increase in T(4) secretion over the control TSH group on the second day, indicating that, in the previous experiments, HC had enhanced pituitary secretion of TSH, rather than the response of the thyroid to TSH . In vivo, 5 days of injections of 10 micrograms HC increased plasma T(4) at prometamorphosis and decreased it at climax . There was no marked effect of HC on plasma or ocular melatonin levels . The findings showed that the nature of the effect of HC on the thyroid axis changes during metamorphosis from inhibition at early stages to a positive influence at prometamorphosis and finally to a negative effect on the T(4) level in the plasma at climax .

J Endocrinol, 1999 Nov, 163(2), 353 - 61
Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes; Wheelhouse NM et al.; Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver . As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets being associated with reduced circulatory IGF-I levels . This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep . Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay . In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0xor 5.0xamino acid media (P<0.01) . Although GH appeared to increase IGF-I release in this period for cells grown in 0.2xamino acid media, this effect was not statistically significant . In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P<0.05 and P<0.001 respectively) . Factorial analysis of variance demonstrated a strong positive interaction (P<0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0xamino acid media (P<0.01) but had no effect on production by cells grown in 1.0xor 0.2xamino acid media.Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level.Incorporation of (35)S-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0xcompared with 5.0x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2xamino acid media (P<0.01) . The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms.Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBPbeta) isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) were determined by Western blotting . When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P<0.05).These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres . The association between these effects and altered levels of C/EBPbeta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.

World J Surg, 1999 Dec, 23(12), 1272 - 8; discussion 1278
Effects of early eschar excision en masse at one operation for prevention and treatment of organ dysfunction in severely burned patients; Huang Y et al.; We sought to determine whether early eschar excision en masse (EEE) at one operation would be effective in the prevention and treatment of postburn organ dysfunction (OD) and multiple organ dysfunction syndrome (MODS) . A total of 60 patients, with total body surface burned area over 35% and a third degree burn area over 20% were studied and divided into two groups, the EEE group (35 cases) and the group treated with repeated escharectomies by stages (repeated escharectomy group, 25 cases) . Other than the different operations undertaken, the patients in both groups received identical conventional treatment . Before, during, and after operation the hemodynamic and blood gas indices, plasma levels of endotoxin and tumor necrosis factor (TNF), and the injurious effects of burn patients' sera on endothelial cells in vitro were determined in patients of the EEE group . The incidence of OD and MODS was decreased significantly (11.4%) in patients of the EEE group, and the cure rate increased greatly (85.7%) . The cardiac output dropped to 77.8% of its preoperative level at the end of escharectomy but began to rise at 2 hours and returned to its baseline levels at 24 hours after operation . Plasma levels of endotoxin and TNF and levels of lactic dehydrogenase and 6-keto-prostaglandin F(1|ga) in the endothelial cell culture media were all reduced profoundly . The cultured endothelial cells maintained their original morphology . The findings substantiate the hypothesis that eschar excision en masse at one operation is feasible and effective in preventing and treating early postburn OD and MODS, mainly by alleviating systemic inflammatory response syndrome and endothelial cell injury.

Jpn J Cancer Res, 1999 Sep, 90(9), 942 - 50
Shedding of membrane type 1 matrix metalloproteinase in a human breast carcinoma cell line; Harayama T et al.; Membrane type 1 matrix metalloproteinase (MT1-MMP) with a transmembrane domain is a new member of the MMP gene family and is expressed on the cell surfaces of many carcinoma cells to activate the zymogen of MMP-2 (gelatinase A) . We have previously reported that MT1-MMP is released into culture media in a complex form with tissue inhibitor of metalloproteinases 2 (TIMP-2) from a human breast carcinoma cell line, MDA-MB-231, treated with concanavalin A (Con A) . In the present study, we further studied the release mechanism of MT1-MMP . Immunoblot analysis indicated that the amounts of MT1-MMP in culture media increase with the time of exposure and the concentration of Con A, and those in cell lysates conversely decrease in a similar way . Time- and dose-dependent release of MT1-MMP into the media was confirmed by a sandwich enzyme immunoassay specific to MT1-MMP . The molecular weight of the immunoreactive MTI-MMP in the media was Mr 56,000, which was 4,000-Mr smaller than that in the cell lysates . Northern blot analysis demonstrated that the mRNA expression level of MT1-MMP is about 3-fold enhanced after a 24 h-exposure to Con A and this is maintained up to 72-h exposure . The release of MT1-MMP from the Con A-treated cells was inhibited by metalloproteinase inhibitors such as EDTA and o-phenanthroline, but not by MMP inhibitors including TIMP-1, TIMP-2 and BB94 or other proteinase inhibitors of serine, cysteine and aspartic proteinases . During the Con A treatment of the cells, cell viability decreased time- and dose-dependently and dead cells reacted positively in the TdT-mediated dUTP Nick-End Labeling (TUNEL) method . Con A-treated MDA cells showed apoptotic morphology when stained with Hoechst dye and hematoxylin and eosin . DNA ladder formation was detected by electrophoresis of the DNA from Con A-treated MDA cells . These results suggest that MT1-MMP release from Con A-treated cells is due to shedding mediated by metalloproteinase(s) other than MMPs, and is associated with apoptosis.

Z Orthop Ihre Grenzgeb, 1999 Sep-Oct, 137(5), 386 - 92
{Current status of autologous chondrocyte transplantation}; Schneider U et al.; PURPOSE: Autogenous chondrocyte transplantation (ACT) is a promising but disputable new method for the treatment of full thickness hyaline cartilage defects . Neither long-term follow-up studies nor prospective randomised comparative clinical trials exist to measure the outcome . METHODS: Several bioengeneering companies have emerged (Genzyme, Verigen, Co.don, etc.) and commercialised this new method of ACT which has generated enormous interest . Each company uses its own (secret) protocol for culturing human cartilage cells . These protocols vary considerably from each other with regard to culture media, enzymes and other additives (e.g . antibiotics) used . However, no quality control requirements for the culturing process of cartilage cells have been proposed which the companies have to fulfill . At the time of surgery the treating surgeon has to assume (and hope) that the cultured cells are viable, sterile, active and potent but a control mechanism does not exist . RESULTS: Considering the enormous price for ACT which the patients and the insurance companies are asked to pay quality control standards should be developed with the following informations given: cell viability, grade of differentiation, cell morphology, secretion levels of essential matrix components (collagen type II, IX, X and XI, Aggrecan) and the total cell number . None of the above information is currently provided by any of the companies involved . CONCLUSION: The widespread clinical use of this technique cannot be recommended at this stage as the scientific proof for reproducibility of ACT has not been given so far . The operative technique of ACT is demanding and should only be used by surgeons having attended special practical training courses and workshops to minimise technical failure rates . Clinical results should be documented in a uniform standardised way using established outcome scores and modern diagnostic measures (e.g . intraoperative biomechanical testing of repair tissue) . Further controlled clinical trials, experimental research and the establishment of quality control standards are required.

Biochem J, 1999 Nov 15, 344 Pt 1, 61 - 8
Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro; Little CB et al.; The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in vitro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration . Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-1 or tumour necrosis factor-alpha . Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay . Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N- and C-terminal neoepitopes generated by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD) . In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken . These analyses indicated that all three catabolic agents stimulated the release of >80% of the GAG from the articular cartilage over 4 days . The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage . Importantly, there was no evidence for the release of MMP-generated aggrecan metabolites into the medium, nor the accumulation of MMP-generated catabolites within the tissue in these same cultures . Expression of the mRNAs for two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP-13, was detected . However, increased expression of these MMPs was not correlated with aggrecan degradation . Analyses using porcine cartilage, cultured with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture . Cultures of late-stage OA human articular cartilage samples indicated that aggrecanase activity was upregulated in the absence of catabolic stimulation when compared with normal porcine or bovine cartilage . In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage . This study demonstrates that the release of aggrecan from both normal and OA cartilage in response to catabolic stimulation in vitro involves a primary cleavage by aggrecanase and not MMPs.

J Nutr, 1999 Nov, 129(11), 2055 - 60
Chronic feeding of a low boron diet adversely affects reproduction and development in Xenopus laevis; Fort DJ et al.; The aims of this work were as follows: 1) to determine whether a purified diet currently used for studies with rats was acceptable for reproductive studies in frogs; and 2) to determine whether frogs are sensitive to a deficit of boron (B) in the diet . Adult Xenopus laevis were fed a nonpurified beef liver and lung (BLL) diet (310 microg B/kg), a purified diet supplemented with boron (+B; 1850 microg B/kg), or a purified diet low in boron (-B; 45 microg B/kg) for 120 d . Frogs fed the BLL and +B diets produced 11.3 and 12.2% necrotic eggs, respectively . Abnormal gastrulation occurred in <4% of the fertilized eggs in both groups, and 96-h larval survival exceeded 75% in both groups . In contrast, frogs fed the -B diet for 120 d produced a high proportion of necrotic eggs (54%) . Fertilized embryos from the -B diet-fed frogs showed a high frequency of abnormal gastrulation (26.8%), and >80% of the embryos died before 96 h of development . Mean embryo cell counts at X . laevis developmental stage 7.5 (mid-blastula) were significantly lower in the -B embryos than in the BLL or +B embryos . BLL and -B embryos grown in low boron culture media had a high frequency of malformations compared with embryos grown in boron-supplemented media . These studies show that a purified diet that has been used in rodent studies was acceptable for reproduction studies in X . laevis . This work also demonstrates that a diet low in boron markedly impairs normal reproductive function in adult X . laevis, and that administration of the low boron diet results in an increase in both incidence and severity of adverse effects . In addition, these studies demonstrate the usefulness of the X . laevis model in nutrition studies.

J Nutr, 1999 Nov, 129(11), 1984 - 91
Secretion of phospholipid transfer protein by human hepatoma cell line, Hep G2, is enhanced by sodium butyrate; Guo Z et al.; Hep G2 cells were used to study the synthesis and secretion of phospholipid transfer protein (PLTP) . Upon incubation of the cells at confluence with serum-free Dulbecco's modified Eagle's medium (DMEM), phosphatidylcholine (PC) transfer activity was found to accumulate in the culture media . The PC transfer activity in the media was effectively inhibited by rabbit anti-human PLTP immunoglobulin (Ig)G, thus indicating that the PC transfer activity was due to secreted PLTP . The molecular weight of Hep G2 PLTP was approximately 78 kDa by Western blot analysis, in agreement with the molecular weight obtained for purified human plasma PLTP . The PLTP secreted by Hep G2 also possessed an HDL conversion activity similar to that of human plasma PLTP . The addition of butyrate to the cell culture media resulted in a marked increase in the secretion of PLTP . After 24 h incubation with 4 mmol/L sodium butyrate, a more than twofold increase (P < 0.01) of PC transfer activity in the cell-conditioned media was obtained . The dose-dependent increase in the PC transfer activity in the media upon butyrate treatment was well correlated (r = 0.80, P < 0.01) with that of PLTP mass as determined by immuno-slot blot analysis of cell-conditioned media . The increased secretion of PLTP by Hep G2 treated with sodium butyrate was accompanied by a greater increase in the level of PLTP mRNA in the cells as determined by ribonuclease protection assay . In the presence of 4 mmol/L sodium butyrate, a fourfold increase (P < 0 . 01) in mRNA level was obtained at 24 h . No stabilizing effect of butyrate on PLTP mRNA was apparent upon treatment of the cultured cells with the RNA synthesis inhibitor, actinomycin D . Thus, the up-regulatory effect of butyrate on PLTP gene expression seemed to have occurred at the transcriptional level.

Cell Prolif, 1999 Apr-Jun, 32(2-3), 131 - 40
Purine nucleotides modulate proliferation of brown fat preadipocytes; Wilson SM et al.; The hypothesis that purine nucleotides and nucleosides affect brown fat preadipocyte proliferation was tested using isolated rat interscapular brown fat preadipocytes in culture . Daily addition of 100 microM adenosine triphosphate (ATP) (n = 4) to cultures enhanced the relative DNA content by 1.5-fold compared to control cultures (P < 0.05) measured using CyQUANT-GR fluorescence . Higher concentrations of ATP inhibited growth and 500 (n = 2) or 1000 microM ATP (n = 3) almost completely inhibited growth . ATP (100 microM) did not affect while 250-1000 microM ATP decreased protein content relative to control cultures . Adenosine (100 microM; n = 3) did not affect DNA or protein content, but 500 microM and 1000 microM adenosine suppressed brown adipocyte proliferation and inhibited protein synthesis . Cultured brown adipocytes quickly removed or degraded ATP in the culture media as determined by luciferin-luciferase bioluminescence, suggesting that the inhibitory effects of high ATP concentrations may result from its breakdown to adenosine . The results support the conclusion that ATP promotes and adenosine inhibits brown adipocyte proliferation.

Physiol Res, 1999, 48(2), 119 - 28
Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells; Vranova J et al.; Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes . The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC) . SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC) . Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC . S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC . L-NAME had no effect on progesterone accumulation by SGC . The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M) . Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone . These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 338 - 44
Improved process for production of recombinant yeast-derived monomeric human G-CSF; Bae CS et al.; The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae . However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction . It was hardly monomerized even by urea at high concentration . This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process . When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures . Also, the monomer fraction and secreted efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5) . Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography . A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents.

Hum Reprod, 1999 Oct, 14(10), 2575 - 80
Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer; Gardner DK et al.; The effect of macromolecules on mouse embryo development and viability after culture in sequential media was investigated . It was found that high rates of viable blastocysts could be obtained in the absence of any macromolecule . Blastocyst cell numbers were increased when bovine serum albumin was present in the culture medium, although this benefit was not manifest after blastocyst transfer . Rather, the highest rates of implantation and fetal development after blastocyst transfer were observed when hyaluronan was the macromolecule in the culture media . Subsequent analysis revealed that the beneficial effects of hyaluronan were due to its presence in the transfer medium . As the highest cell numbers and hatching rates obtained in this study occurred when both serum albumin and hyaluronan were present in the same medium, it is proposed that embryo culture media should contain both serum albumin and hyaluronan, while the transfer medium need only contain hyaluronan.

Hum Reprod, 1999 Oct, 14(10), 2525 - 30
Dimeric inhibins and activin A in human follicular fluid and oocyte-cumulus culture medium; Lau CP et al.; The concentrations of inhibin A, inhibin B and activin A in follicular fluid and oocyte culture medium were analysed to investigate the production of these peptide hormones by ovarian granulosa cells and oocyte-cumulus complexes, as well as their potential as possible biochemical markers for oocyte quality and fertilizing capacity . Follicular fluids were collected from individual follicles during oocyte retrieval for in-vitro fertilization (IVF) . Oocyte-cumulus culture media were collected after in-vitro insemination . The concentrations of dimeric inhibin A, inhibin B and activin A were measured using two-site enzyme-linked immunosorbent assays in the follicular fluid and matched oocyte culture medium . Hormone concentrations were compared with oocyte quality and fertilizing capacity . The concentration of inhibin A in follicular fluid increased while that of inhibin B decreased with increasing follicle size . Follicular fluid concentrations of inhibin A inhibin B and activin A were not significantly different in follicles with differing oocyte quality . Oocyte culture medium concentrations of activin A were significantly higher in morphologically good quality oocytes . There was no relationship between the concentrations of the three hormones and oocyte fertilizing capacity . This study confirms that follicular fluid concentrations of inhibin A may prove to be a marker of follicular growth and maturation . Higher concentrations of activin A produced by good quality oocyte-cumulus complexes suggest that activin A may play a role in oocyte maturation.

Pharmacol Res, 1999 Nov, 40(5), 423 - 7
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) inhibits nitric oxide production in cultured murine astrocytes; Jung YD et al.; The level of nitrite accumulation in the culture media of astrocytes activated with lipopolysaccharide (LPS) and interferon-gamma (IFN) was decreased by pretreatment of cells with LY294002, a quercetin derivative developed for phosphatidylinositol-3-kinase (PI3K) inhibitor, in a dose-dependent manner . The expression of iNOS mRNA in the astrocytes was inhibited by LY294002, as revealed by reverse transcriptional polymerase chain reaction and agarose gel electrophoresis . The catalytic activity of astrocytic iNOS was also inhibited by LY294002 . On the other hand, wortmannin which was known to enhance endotoxin-induced NO production in macrophages by inhibiting PI3K did not cause any significant change in the NO production and iNOS mRNA expression of the astrocytes . These results suggest that LY294002 suppresses NO production in the astrocytes through not only the inhibition of iNOS mRNA expression but also the inhibition of the iNOS activity and that PI3K is not involved in the inhibitory actions of LY294002.pc 1999 Academic Press@p$hr

Blood Rev, 1999 Sep, 13(3), 171 - 84
Perfluorinated blood substitutes and artificial oxygen carriers; Lowe KC; Blood transfusion is a remarkably safe, routine clinical procedure . However, the need for sophisticated blood processing, storage and cross-matching, coupled with increasing concerns about the safety of blood products, has fuelled the search for safe and efficacious substitutes . Candidate materials based on modified haemoglobin (including recombinant molecules) or highly inert, respiratory gas-dissolving perfluorinated liquids (perfluorochemicals) have been developed . The latter are immiscible in aqueous systems and must, therefore, be injected as emulsions . Second-generation perfluorochemical emulsions are available and in clinical trials as temporary intravascular oxygen carriers during surgery, thereby reducing patient exposure to donor blood . One commercial product is currently under Phase III clinical evaluation, with regulatory approval expected within 1 2 years . Other biomedical applications for perfluorochemicals and their emulsions include their use as pump-priming fluids for cardiopulmonary bypass, lung ventilation fluids, anti-cancer agents, organ perfusates and cell culture media supplements, diagnostic imaging agents and ophthalmologic tools . Novel applications for perfluorochemicals as immunomodulating agents are also being explored.

Diabetologia, 1999 Oct, 42(10), 1219 - 27
Amyloid fibril formation is progressive and correlates with beta-cell secretion in transgenic mouse isolated islets; MacArthur DL et al.; AIMS/HYPOTHESIS: Amyloid fibrils are formed in islets isolated from transgenic mice expressing the gene for human islet amyloid polypeptide (IAPP) by an unknown mechanism . This model of islet amyloidosis in Type II (non-insulin-dependent) diabetes mellitus has been used to investigate the temporal and glucose dependency of fibril formation . METHODS: To determine the time course and nature of amyloid-like accumulations and the role of glucose, transgenic mouse islets were cultured for 2-12 days in medium containing glucose (4.2 mmol/l, 11.1 mmol/l or 16.7 mmol/l) or 3.3 mmol/l glucose plus non-glucose secretagogues, 10 mmol/l leucine, 10 mmol/l leucine + 0.1 mmol/l tolbutamide, 10 mmol/l alpha-ketoisocaproic acid + 10 mmol/l glutamine . The extent of fibril formation was determined by quantitative immuno-electron microscopy . Insulin and islet amyloid polypeptide secretion into the media was measured by radioimmunoassay . RESULTS: Extracellular amyloid fibrils immunoreactive for islet amyloid polypeptide were visible initially after 6 days of culture in 11.1 mmol/l glucose and formed 2.3 +/- 0.8 % of the islet area after 12 days; small accumulations of intracellular fibrils and amorphous extracellular islet amyloid polypeptide-immunoreactive material were present at 6-12 days . Beta-cell secretion was increased significantly by 16.7 mmol/l glucose and by alpha-ketoisocaproic acid + glutamine . The proportion of fibrillar amyloid (amyloid area/islet area%) correlated with the amount of insulin (r = 0.55, p < 0.05) and IAPP (r = 0.5, p < 0.05) in the culture media . Evidence of cellular damage was present in less than 10 % cells and correlated with the degree of fibril deposition (r = 0.8, p < 0.0001) . CONCLUSION/INTERPRETATION: These data suggest that islet amyloid polypeptide amyloid is formed primarily at extracellular sites in isolated transgenic mouse islets and progressive fibril formation correlates with beta-cell secretion . {Diabetologia (1999) 42: 1219-1227}

Amino Acids, 1999, 17(2), 165 - 73
Effect of some herbicides on the production of lysine by Azotobacter chroococcum; Gonzalez-Lopez J et al.; Production of lysine by Azotobacter chroococcum strain H23 was studied in chemically-defined media amended with different concentrations of alachlor, metolachlor, 2,4-D, 2,4,5-T and 2,3,6-TBA . The presence of 5, 10, and 50 micrograms/ml of alachlor or 2,3,6-TBA significantly decreased quantitative production of lysine . However, the presence 2,4-D or 2,4,5-T at concentrations of 10 and 50 micrograms/ml enhanced the production of lysine . Quantitative production of lysine was not affected as consequence of the addition of metolachlor to the culture medium, showing that the release lysine to the culture media by A . chroococcum was not affected by that herbicide.

Gene, 1999 Sep 3, 237(1), 153 - 9
Improved assay sensitivity of an engineered secreted Renilla luciferase; Liu J et al.; We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2 . The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity . In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues . Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity . Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase . This increase in light emission was due in part to enhanced stability of the mutant enzyme . This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.

Inflamm Res, 1999 Sep, 48(9), 497 - 502
The beta-adrenoceptor agonist clenbuterol is a potent inhibitor of the LPS-induced production of TNF-alpha and IL-6 in vitro and in vivo; Izeboud CA et al.; OBJECTIVE AND DESIGN: To investigate the suppressive effects of the beta-agonist clenbuterol on the release of TNF-alpha and IL-6 in a lipopolysaccharide (LPS)-model of inflammation, both in vitro and in vivo . MATERIAL AND SUBJECTS: Human U-937 cell line (monocyte-derived macrophages), and male Wistar rats (200-250 g) . TREATMENT: U-937 macrophages were incubated with LPS at 1 microg/ml, with or without 1.0 mM-0.1 nM test drugs (clenbuterol and other cAMP elevating agents) for 1-24 h . Rats were administered either 1 or 10 microg/kg clenbuterol (or saline) orally, 1 h before intraperitoneal administration of 2 mg/kg LPS . METHODS AND RESULTS: TNF-alpha and IL-6 time-concentration profiles were determined both in culture media and plasma, using ELISA' s and bioassays . LPS-mediated release of both cytokines was significantly suppressed by clenbuterol . CONCLUSIONS: The beta-agonist clenbuterol very potently suppresses the LPS-induced release of the pro-inflammatory cytokines TNF-alpha and IL-6 both in vitro and in vivo.

FEBS Lett, 1999 Sep 10, 458(1), 23 - 6
Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells; Tokuyasu K et al.; With the aid of a signal sequence of a chitinase from Streptomyces lividans, a recombinant chitin deacetylase, whose gene originated from a Deuteromycete, Colletotrichum lindemuthianum, was produced in the culture medium of Escherichia coli cells, existing as a highly active form without the signal peptide . During the production of the recombinant chitin deacetylase, both a slight increase in the value of OD600 nm in the culture medium and a drastic decrease in viable cell number were observed . When penta-N-acetyl-chitopentaose was used as the substrate, the recombinant chitin deacetylase had comparable kinetic parameters to those of the original enzyme from the fungus . The addition of a C-terminal six histidine sequence to the recombinant enzyme caused a slight decrease in the kcat value, and the further addition of a 12 amino acid sequence at its N-terminus caused a further decrease in the value . This production system allowed us to easily produce in the culture media the recombinant chitin deacetylases possessing as good properties as the original enzyme, without any disruption steps of the E . coli cells.

Matrix Biol, 1999 Aug, 18(4), 343 - 55
beta-Integrin-collagen interaction reduces chondrocyte apoptosis; Cao L et al.; We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis) . Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte-matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis . In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival . Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis . Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation . Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis . Experiments with an antibody to integrin beta1 suggest that the collagen-chondrocyte interactions are mediated through integrin beta1, and these interactions may protect chondrocytes from apoptosis . We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte-matrix network.

Ophthalmologica, 1999, 213(5), 311 - 9
The effect of hydroxyurea on rabbit subconjunctival fibroblast culture and use of hydroxyurea in rabbits after glaucoma filtration surgery; Yucel I et al.; In an in vitro study, rabbit subconjunctival fibroblasts were cultured and the effects of an antineoplastic drug, hydroxyurea (HU), on fibroblast proliferation and fibroblast attachment was investigated . The effects of HU were compared with those of mitomycin C (MMC) . Different concentrations of HU and MMC were added to culture medium . The HU doses which led to 50% of inhibition (ID(50)) and the dose which led to about 90% of inhibition (subtoxic high dose, STHD) were determined to be 8 and 1,000 microg/ml, respectively . ID(50) of MMC and its STHD which led to about 100% inhibition were found to be 0.01 and 1 microg/ml, respectively . Reversibility studies revealed that inhibition disappeared depending on the dose and incubation period of both HU and MMC . In an in vivo study, glaucoma filtration surgery (GFS) was performed in rabbits which were treated with HU (treatment group) and distilled water (control group) . Tissue samples were taken from the subconjunctival area treated at 1 h, 1 day, 5 days and 30 days postoperatively . The biopsy specimens were then placed in tissue culture media . Fibroblast outgrowth rates detected in the HU group were found to be significantly lower than those in the control group in the specimens taken at the end of the first hour . The difference was significant on culture days 9-15 in the biopsy specimens taken on day 1 while it was not significant in those taken on days 5 and 30.

Invest Ophthalmol Vis Sci, 1999 Oct, 40(11), 2652 - 9
Induction of glutathione S-transferase hGST 5.8 is an early response to oxidative stress in RPE cells; Singhal SS et al.; PURPOSE: To delineate the role of the glutathione S-transferase (GST) isozyme hGST 5.8 in protection mechanisms against oxidative stress, the effect of low-level transient exposure of H2O2 to retinal pigmented epithelial (RPE) cells on hGST 5.8 and other enzymes involved in defense against oxidative stress was examined . METHODS: Cultured human RPE cells were exposed to 50 microM H2O2 for 20 minutes . Subsequently, the cells were washed and resuspended in the culture media . The cells were pelleted and lysed, and the levels of lipid peroxidation products including thiobarbituric acid-reactive substances (TBARS), glutathione (GSH), glutathione peroxidase (GPX), glucose 6-phosphate dehydrogenase, glutathione reductase, GST, catalase (CAT), and superoxide dismutase (SOD) were determined and compared with levels in control cells . Total GSTs were purified by GSH-affinity chromatography, and the isozymes were separated by isoelectric focusing, characterized, and quantitated . hGST 5.8 was quantitated by an immunologic method as well as by determining activity toward its preferred substrate, 4-hydroxynonenal (4-HNE) . Kinetic constants of hGST 5.8 purified from H2O2-treated cells were also determined and compared with those of control cells . RESULTS: Exposure of RPE cells to 50 microM H2O2 for 20 minutes showed a significant increase in TBARS (1.8-fold) and gamma-glutamyl cysteine synthetase (gamma-GCS) activity (1.6-fold) . A significant increase (1.2-fold) was also observed in GPX activity toward cumene hydroperoxide, but CAT and SOD activities remained unchanged . There was no significant increase in GST activity toward 1-chloro-2, 4-dinitrobenzene but GST activity toward 4-HNE was increased by 1.4- to 1.8-fold . The increase in GST activity toward 4-HNE was associated with a 2.8-fold increase in protein of the isozyme hGST 5.8, which uses 4-HNE as the preferred substrate . CONCLUSIONS: Results of these studies show that the induction of hGST 5.8, which is involved in the detoxification of the lipid peroxidation products 4-HNE and hydroperoxides, may be an early adaptive response of RPE cells exposed to low levels of transient oxidative stress . It is suggested that this isozyme may be crucial for protecting the RPE from low levels of chronic oxidative stress . Observed increases in GPX and gamma-GCS activities are consistent with this idea, because GPX activity is also expressed by hGST 5.8, and gamma-GCS is the rate-limiting enzyme in biosynthesis of GSH, the substrate for hGST 5.8.

Pediatr Res, 1999 Oct, 46(4), 406 - 10
Mechanism for dexamethasone inhibition of neutrophil migration upon exposure to lipopolysaccharide in vitro: role of neutrophil interleukin-8 release; Zentay Z et al.; The mechanism by which dexamethasone (DEX) inhibits neutrophil (PMN) recruitment to a site of inflammation, such as the newborn lung with bronchopulmonary dysplasia, is not completely understood . The aim of our study was to determine whether DEX inhibits neutrophil-induced neutrophil recruitment by inhibition of interleukin- (IL) 8 release from PMNs, and if there are developmental differences . PMNs isolated from cord blood (CB) and adults (A) were studied . We first measured the effect of DEX (10(-10) to 10(-4) M) on PMN migration to an exogenous IL-8 standard (10(-8) M) using PMNs of CB (n = 3) and A (n = 3), over 1 h in a chemotaxis chamber . Second, we determined the effect of DEX (0 and 10(-10) to 10(-6) M) on IL-8 release (immunoassay) from PMNs of CB (n = 7) or A (n = 7) after incubation with lipopolysaccharide (LPS, 1 ng/mL) for 6 and 18 h . Third, the chemoattractant activity of culture media from the second experiment was studied with and without IL-8 antibody . DEX at concentrations of 10(-10) to 10(-4) M had no direct effect on PMN migration in vitro to an exogenous IL-8 standard . After LPS exposure, IL-8 release was greatly increased for PMNs from CB compared with A . DEX (10(-10) to 10(-4) M) resulted in a dose-dependent inhibition of IL-8 release from PMNs exposed to LPS for 6 and 18 h incubation . Increased PMN migration activity was only found with media of PMNs of CB with no DEX . At 18 h, media-induced migration activity was decreased if DEX (10(-7) M), IL-8 antibody, or DEX (10(-7) M) with IL-8 antibody were present during the incubation with LPS: there was an 88, 86, and 101% reduction in migration activity, respectively . We conclude that DEX inhibits PMN-induced PMN migration, predominantly via inhibition of IL-8 release for PMNs of the newborn . We suggest that a 10-fold lowering of the standard DEX dose may effectively reduce lung inflammation in bronchopulmonary dysplasia.

Neurosci Lett, 1999 Aug 27, 271(3), 179 - 82
Characterization of rat spinal cord neurons cultured in defined media on microelectrode arrays; Manos P et al.; Previous efforts to utilize mammalian spinal cord neurons as biosensor elements have relied on neuronal: glial co-cultures maintained in serum-containing media . We have examined the feasibility of culturing primary spinal cord neurons in serum-free medium, modified for neuronal longevity, on fabricated microelectrode arrays . Embryonic day 15 rat spinal cord cells were plated on trimethoxysilyl-propyldiethylenetriamine coated microelectrode arrays comprised of gold recording sites passivated with silicon nitride . Immunocytochemistry was performed to verify the presence of neurons and quantitatively assess astrocytes using antibodies against glial fibrillary acidic protein on the silicon nitride substrates . Modifications to culture media enabled viable neuronal culture to extend from approximately 14 days in vitro (DIV) to 40 DIV on the arrays containing only 1.1 +/- 0.5% (mean +/- SEM) astrocytes . Extracellular recording revealed tetrodotoxin-sensitive spontaneous electrical activity from the enriched neuronal culture . Threshold detection of extracellular potentials showed an increase in spike rate as a function of glutamate concentration with neurotoxicity at elevated levels . This approach suggests that functional measures related to biosensor applications, pharmacological screening, or the evaluation of neurological disease models can be implemented in a defined culture system.

Ann Clin Biochem, 1999 Sep, 36 ( Pt 5), 660 - 5
Expression and secretion of neural cell adhesion molecules by human pituitary adenomas; De Jong I et al.; Neural cell adhesion molecules (NCAMs) are found predominantly in neural, muscle and endocrine cells . Recent interest has focused on their potential role in tumorigenesis . We have analysed the expression and secretion of NCAM in a series of 48 human pituitary adenomas . Immunocytochemical analysis of 19 adenomas demonstrated NCAM expression in all tumours with, in each case, diffuse cytoplasmic staining being found with variable membrane accentuation . There were no apparent differences in the expression of immunoreactivity seen on sections between individual tumours . Cell culture media from 43 dispersed human pituitary tumours were analysed by immunoassay for the secretion of soluble NCAM and all the pituitary hormones . In contrast to the immunocytochemical studies, soluble NCAM was released from only 27% of human pituitary tumours, but this was not related to tumour type nor was the amount of soluble NCAM released correlated with the amount of pituitary hormone secreted by each adenoma . NCAM expression is common to all human pituitary adenoma types and the observed differences in release of soluble NCAM between individual tumours may reflect different molecular mechanisms, altering adhesive interactions between normal and adenomatous tissue.

Kidney Int, 1999 Oct, 56(4), 1366 - 77
Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells; Leonard M et al.; BACKGROUND: Both interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic cytokines that have been implicated in the development of glomerular and tubular injury in various forms of immune-mediated renal disease, including glomerulonephritis . Although TNF-alpha has been shown to stimulate IL-6 production in renal cells in culture, the signaling mechanisms that regulate IL-6 production are not fully understood . The aim of this study was to examine the role of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways in regulating TNF-alpha-mediated IL-6 production from both primary human mesangial cells (HMCs) and human proximal tubular (HPT) cells . METHODS: Primary mesangial and proximal tubular cells were prepared from nephrectomized human kidney tissue . Cells were treated for 24 hours with TNF-alpha in the presence and absence of the specific p38 and ERK1,2 MAPK inhibitors SB203580 and PD98059, respectively, either alone or in combination . IL-6 levels in the cell culture media were measured by enzyme-linked immunosorbent assay . MAPK activation was demonstrated by immunoblot for the active kinase (tyrosine/threonine phosphorylated) in whole cell extracts using phospho-specific antibodies . p38 MAPK activity in HPT cells was measured using an in vitro immunokinase assay using ATF2 as the substrate . RESULTS: TNF-alpha (0.1 to 100 ng/ml) stimulated a dose-dependent increase in IL-6 production in both renal cell types . The activation of the p38 and the ERK1,2 MAPKs occurred following TNF-alpha stimulation . The role of these activations in IL-6 production was confirmed by the ability of both inhibitors SB203580 (1 to 30 microM) and PD98059 (0.01 to 10 microM) to inhibit basal and TNF-alpha-stimulated IL-6 production in both cell types . The addition of both inhibitors in combination caused greater decreases in IL-6 production compared with either inhibitor alone . Pretreatment with SB203580 (10 microM) had no effect on basal or TNF-alpha-stimulated phosphorylation of p38 MAPK but completely abolished TNF-alpha-stimulated p38 MAPK activity . PD98059 decreased both basal and TNF-alpha-stimulated phosphorylation of ERK1,2 . CONCLUSIONS: This study provides evidence that both the p38 and ERK MAPK pathways are important for the regulation of the production of IL-6 from the proximal tubular and glomerular mesangial regions of the nephron . In response to TNF-alpha, the activation of both pathways leads to IL-6 production . These findings could aid in an understanding of the cellular mechanisms that regulate IL-6 production and could provide insights into possible pharmacological strategies in inflammatory renal disease.

Endocr J, 1999 Jun, 46(3), 389 - 96
Establishment of an estrogen responsive rat pituitary cell sub-line MtT/E-2; Fujimoto N et al.; A rat pituitary tumor sub-line MtT/E-2 was established from a rat pituitary tumor cell line MtT/E . Its growth was found to depend on the presence of estradiol (E2) in culture media at 10(-13)-10(-9) M, whereas the original cell line MtT/E proliferated autonomously . The recently discovered PTTG (pituitary tumor transforming gene) is highly expressed in this cell line, although not regulated by E2 . On the other hand, E2 induced c-myc and cyclin D1 proteins in MtT/E-2, which contains a lot (220+/-18 fmol/mg protein) of estrogen receptor demonstrated by RT-PCR analysis to be predominantly alpha type . MtT/E-2 secretes growth hormone which is, interestingly, regulated by retinoic acid and dexamethasone rather than thyroid hormones.

J Biochem (Tokyo), 1999 Oct, 126(4), 694 - 9
A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase; Oda K et al.; A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP) . To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA . The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric . The enzyme was used for production of antibodies specific for human TNSALP . Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells . However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides . The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.

J Cell Biochem, 1999 Nov 1, 75(2), 206 - 14
Inhibitory effects of activin-A on osteoblast differentiation during cultures of fetal rat calvarial cells; Ikenoue T et al.; Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts . However, the role of activin-A on osteoblasts is not clearly understood . We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones . Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay . This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture . In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis . The peak of osteocalcin mRNA was delayed . Gene expression for TGF-beta was not influenced by exogenous activin-A . The betaA subunit (activin-A) mRNA was detected during the early phase of this culture . These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.

In Vitro Cell Dev Biol Anim, 1999 Sep, 35(8), 465 - 71
The influence of culture media on embryonic renal collecting duct cell differentiation; Schumacher K et al.; During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function . The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells . Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture . The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove's modified Dulbecco's medium as compared to medium supplemented with additional NaCl . Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive . To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams' medium E, McCoys 5A medium, and Dulbecco's modified Eagle medium under serum-free conditions . The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia . According to morphological criteria, there is no difference in morphological epithelial cell preservation . The immunohistochemical data reveal two groups of expressed antigens . Constitutively expressed antigens such as cytokeratin 19, P CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used . In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series . Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.

Adv Exp Med Biol, 1999, 457, 397 - 407
Pharmacokinetics of anticancer drugs in vitro; Wagner A et al.; It is generally assumed that drug concentration does not change significantly under cell culture conditions . Nevertheless, most of the therapeutic trials in acute leukemia that were based on in vitro drug sensitivity assays of patient samples have been disappointing . In order to show possible pitfalls of unphysiological alterations in vitro we investigated concentration versus time curves, metabolism and effects on the culture media for some antineoplastic drugs . Oxazaphosphorines and cytarabine were incubated in RPMI and in established cell lines and measured by HPLC . HPLC also served to measure enzyme activity and levels of related amino acids at various concentrations of asparaginase, ammonia release was photometrically determined . Etoposide was monitored by HPLC relative to different contents of FCS in RPMI . All oxazaphosphorines showed a rapid decrease of in vitro activity down to about 10% within 4-6 h, and 2% within 72 h . The level of cytarabine, when incubated in RPMI, was stable over 24h, and no change was seen with K562, while a rapid decrease to below 50% occurred within 6h in the presence of HL 60 and BLIN . 2 U/L of asparaginase led to asparagine depletion of the medium within 4h, while 200 U/L were associated with a preferential increase of glutamic acid and ammonia . Further, there was evidence of instability by rapid adsorption to plastic surfaces (paclitaxel) or isomerisation (etoposide) in RPMI with low FCS content . The instability of drugs in vitro is attributed to a variety of different factors: i.e . physico-chemical instability results in inactivation of oxazaphosphorines, cytarabine disappears by cellular metabolism without saturation depending on the cell-line . Epiphenomena like adsorption and isomerisation in vitro are unphysiological . Results of drug sensitivity assays should be interpreted with great caution.

Peptides, 1999, 20(9), 1123 - 5
Production and secretion of adrenomedullin in cultured human alveolar macrophages; Nakayama M et al.; To explore the role of adrenomedullin (ADM) in macrophages, we investigated the secretion of ADM by alveolar macrophages . Human alveolar macrophages obtained from bronchoalveolar lavage were cultured for 24 h . Northern blot analysis revealed ADM mRNA expression in alveolar macrophages . The levels of immunoreactive ADM in the media were 0.89+/-0.12 fmol/10(5) cells/24 h (n = 10) . Reverse-phase high-performance liquid chromatography of the extract of culture media showed one major peak eluting in the position of the human ADM standard . The present study shows that alveolar macrophages produce and secrete ADM.

Oncogene, 1999 Sep 16, 18(37), 5167 - 76
Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism; Dorman CM et al.; Chronic overexpression of the oncogenic form of Ras is a potent inhibitor of skeletal myogenesis . However, the intracellular signaling pathways that mediate the repressive actions of Ras on myogenic differentiation have yet to be identified . We examined the role of Raf-mediated signaling as a modulator of avian myogenesis . Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes . Most notably, the embryonic chick myoblasts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or RCAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures . While residual myofibers were apparent in the RCAS-Raf BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic phenotype . The altered morphology is not a result of reinitiation of the myonuclei cell cycle nor is it due to apoptosis . Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity . Supplementation of the culture media with the MAPK kinase (MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls . Our results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to myogenesis.

Toxicol Sci, 1999 Sep, 51(1), 80 - 6
Chemically induced oxidative stress disrupts the E-cadherin/catenin cell adhesion complex; Parrish AR et al.; The impact of xenobiotics on intercellular adhesion, a fundamental biological process regulating most, if not all, cellular pathways, has been sparsely investigated . Cell-cell adhesion is regulated in the epithelium primarily by the E-cadherin/catenin complex . To characterize the impact of oxidative stress on the E-cadherin/catenin complex, precision-cut mouse liver slices were challenged with two model compounds for the generation of oxidative stress, diamide (DA; 25-250 microM) or t-butylhydroperoxide (tBHP; 5-50 microM), for 6 h . At the concentrations used, neither compound elicited cytotoxicity, as assessed by intracellular K+ content and leakage of lactate dehydrogenase into the culture media . However, a 25% reduction in non-protein sulfhydryl levels, an indication of oxidative perturbation, was seen in liver slices treated with DA or tBHP . Total protein expression of E-cadherin, beta-, or alpha-catenin was not affected by challenge with DA or tBHP . A decrease of beta-catenin in the SDS-soluble fraction of slices, an indicator of the formation of the adhesion complex, was observed . Additionally, a decrease in beta-catenin interactions with E-cadherin and alpha-catenin, as assessed by immunoprecipitation and Western blot analysis, was seen . Disruption of the E-cadherin/catenin complex by tBHP, but not DA, correlated with enhanced tyrosine phosphorylation of beta-catenin . These results suggest that noncytotoxic oxidative stress disrupts the E-cadherin/catenin cell adhesion complex in precision-cut mouse liver slices.

Br J Cancer, 1999 Sep, 81(2), 342 - 9
Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer; Takahashi T et al.; Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB) . We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250-256) recognized by human antibodies and overexpressed by breast cancer cells . In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells . These decamers, GLQKASIFLK (247-256) and KASIFLKTRV (250-259), have anchor motifs for both HLA-A2 and HLA-A3 . Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml(-1) interleukin-2, 25 IU ml(-1) interleukin-7 and 500 IU ml(-1) granulocyte-macrophage colony-stimulating factor . Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide . Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors . Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors . No HLA-A2-, A3 CTL line showed specific cytotoxicity against these target cells . These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line . RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer.

J Urol, 1999 Oct, 162(4), 1259 - 63
A feasibility study of gene gun mediated immunotherapy for renal cell carcinoma; Seigne J et al.; PURPOSE: Gene modified autologous tumor cell vaccines have demonstrated a protective and therapeutic effect in murine tumor model systems . The majority of trials to date have used viral methods of gene transfer for vaccine construction . An alternative approach to transfer genes into tumor cells is to use the gene gun, which is a physical method of gene transfection that produces high levels of gene expression without viral agents . We establish the feasibility of generating cytokine secreting autologous renal tumor cell vaccines for use in gene therapy for metastatic renal cell carcinoma . MATERIALS AND METHODS: We obtained 1 cm3 tumor tissue from 12 patients undergoing resection of primary or metastatic renal cell carcinoma . The tumor was disaggregated and placed in culture . The phenotype of the primary renal cell lines was established by microscopy and immunohistochemistry . The 1x10(7) lethally irradiated tumor cells were transfected with plasmid deoxyribonucleic acid containing the human (h) granulocyte-macrophage colony-stimulating factor (GM-CSF) gene under control of a cytomegalovirus promoter using the gene gun . The hGM-CSF production was assayed by enzyme-linked immunosorbent assay in the cell culture media 24 hours after transfection . RESULTS: Of 12 tumor samples 8 grew rapidly to produce a mean of 1.8x10(8) cells after 4 to 5 passages in culture, which was sufficient to produce between 24 and 32 vaccines . Immunocytochemistry confirmed that all cultures were almost exclusively renal tumor cells . Gene gun mediated transfection of lethally irradiated tumor cells resulted in high levels of hGM-CSF production (mean 330 ng./10(6) cells per 24 hours) . CONCLUSIONS: We have demonstrated the feasibility of producing cytokine secreting tumor cell vaccines from primary and metastatic human renal tumors, and plan to use this approach in phase I clinical trials of gene therapy for metastatic renal cell carcinoma.

Mol Cell Biol, 1999 Oct, 19(10), 7096 - 105
Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4; Monzen K et al.; Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart . However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown . In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells . We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin . Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes . The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system . Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation . Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes . These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Exp Neurol, 1999 Sep, 159(1), 237 - 47
Regeneration and proliferation of embryonic and adult rat hippocampal neurons in culture; Brewer GJ; Adult mammalian CNS neurons appear to be terminally differentiated and postmitotic . However, this conclusion may be due to nonpermissive conditions in the brain or in culture media . If embryonic rat hippocampal neurons are cultured in Neurobasal/B27 with FGF2, nearly all neurons proliferated until a maximum density was reached . Similarly, adult neurons can be cultured that fire action potentials and display immunoreactivity for neurofilament, MAP2, tau, and glutamate . Seventy percent of the 3000 isolated adult cells per milligram of brain tissue began to proliferate after 3 days in culture and incorporated BrdU . By 4 days of regeneration in culture, virtually all neuron-like cells with asymmetric processes were glutamate positive and immunoreactive for neurofilament . Immunoreactivity of the intermediate filament stem cell marker nestin increased in adult cells to levels present in freshly isolated embryonic neurons . These are the first studies to demonstrate that over 50% of adult CNS cells with neuron-like characteristics retain regenerative and proliferative potential .

Biotechnol Bioeng, 1999 Nov 5, 65(3), 298 - 305
Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection; Mastrangelo AJ et al.; Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture . In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems . Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy . Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade . In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production . N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells . In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls . Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples . In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases . Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures . Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs .

Neurochem Res, 1999 Sep, 24(9), 1189 - 93
Possible involvement of GABA(A) and GABA(B) receptors in the inhibitory action of lindane on transmitter release from cerebellar granule neurons; Damgaard I et al.; Cerebellar granule cells in culture express receptors for GABA belonging to the GABA(A) and GABA(B) classes . In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 microM THIP as this is known to influence the properties of both GABA(A) and GABA(B) receptors . It was found that lindane regardless of the culture condition inhibited evoked (40 mM K+) release of neurotransmitter ({3H}D-aspartate as label for glutamate) . In naive cells both GABA(A) and GABA(B) receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA(A) and GABA(B) receptor active drugs had any effect on the inhibitory action of lindane . This lack of effect was not due to inability of baclofen itself to inhibit transmitter release . It is concluded that lindane dependent on the state of the GABA(A) and GABA(B) receptors is able to indirectly interfere with both GABA(A) and GABA(B) receptors . In case of the latter receptors it was shown using {3H}baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.

Neurochem Res, 1999 Sep, 24(9), 1107 - 15
Gangliosides attenuate ethanol-induced apoptosis in rat cerebellar granule neurons; Saito M et al.; Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 microM MK-801 (an NMDA receptor antagonist), and 20-200 mM ethanol for 1-4 days . Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3 . Inclusion of 5 microM MK-801 or 100 microM glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway . Preincubation with 50 microM gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 microM LIGA20 (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death . Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.

Exp Clin Endocrinol Diabetes, 1999, 107(5), 281 - 7
The effect of testicular macrophages, macrophage-conditioned medium and interleukin-1alpha on the cytoskeleton of bank vole Leydig cells; Bilinska B et al.; Recently, morphological and functional interactions between cytoskeletal elements and their involvement in cell movements, shape changes and/or translocation of organelles have been intensively studied . Thus, the aim of our work was to determine whether testicular macrophages and/or their products have an influence on Leydig cell cytoskeleton . The source of Leydig cells and macrophages were male bank voles from spring and autumn generations, reared in different regime of light for 7-8 weeks . The Leydig cells were growing in monocultures or in co-cultures with testicular macrophages . All cell cultures were divided to controls or human chorionic gonadotropin-stimulated ones . To some of the cultures testicular macrophage-conditioned medium or interleukin-1alpha were added . The cells were analysed immunocytochemically and radioimmunologically . In Leydig cells obtained from animals kept in a long day, grown in co-cultures with macrophages as well as in those stimulated by testicular macrophage-conditioned medium, distinct rearrangements of microtubules and microfilaments were observed . This phenomenon was strengthened in the presence of hCG in culture media . Concomitantly, basal and hCG-stimulated level of testosterone was enhanced, which indicates the possible involvement of the cytoskeleton in the process of androgen biosynthesis . The influence of IL-1alpha on reorganization of cytoskeletal structures was not observed, suggesting that in the modulation of steroidogenesis by this cytokine cytoskeletal elements do not play an important role.

Prostaglandins Other Lipid Mediat, 1999 Jul, 57(5-6), 361 - 70
Contribution of type II PLA2 to prostaglandin formation: a study using a type II PLA2 specific inhibitor SB 203347; Munns MJ et al.; Arachidonic acid (AA) mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is considered to be a pivotal event in inflammation . The purpose of this study was to assess the efficacy of a Type II PLA2 specific inhibitor, SB 203347, in reducing prostaglandin production in Type II PLA2-transfected Chinese hamster ovary (CHO) cells and in human placenta . In both experimental models utilised, Type II PLA2 represents the principal isozyme contributing to total PLA2 enzymatic activity . PLA2 enzymatic activity released into cell culture media and placental explant media was quantified by radiolabelled substrate assay {14C-phosphatidylethanolamine (PE)} . Immunoreactive prostaglandin F2alpha (PGF2alpha) concentrations were determined by radioimmunoassay . SB 203347 (at 0.1-10 microM final concentration) inhibited PLA2 enzymatic activity released by Zn++ -activated CHO cells by up to 60% (P<0.0001) . The concentration of PGF2alpha present in culture media was concomitantly reduced by up to 90% (P<0.0001) . Similar results were observed for human placental explants . Treatment of human placental explants with SB 203347 (1 microM final concentration) significantly reduced PLA2 enzymatic activity recovered in media after 24 h incubation (P<0.0001; n = 10) . Incubation media PGF2alpha concentrations were also reduced by 60% (P<0.00001) . The addition of endogenous arachidonic acid (30 microM final concentration) significantly attenuated SB 203347-inhibition of PGF2alpha release (P<0.01) . The data obtained in this study are consistent with the hypothesis that Type II PLA2 contributes to the liberation of arachidonic acid for prostanoid formation in human placenta and in cells that abundantly express this isozyme.

Peptides, 1999, 20(6), 769 - 72
Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells; Nakayama M et al.; To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells . Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively . The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM) . Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard . The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia . ADM may therefore play an important pathophysiological role in ischemic heart disease.

In Vitro Cell Dev Biol Anim, 1999 Jun, 35(6), 346 - 51
Human endothelial cells cultured on microporous filters used for leukocyte transmigration studies form monolayers on both sides of the filter; Mackarel AJ et al.; A growing number of studies on the mechanism of leukocyte transendothelial migration use endothelial cells grown on microporous filters as an in vitro model of endothelium . Ultrastructural examination of such a model system previously demonstrated that human pulmonary artery endothelial cells (HPAEC) formed confluent monolayers on both sides of the 3-microm-pore filter (Mackarel et al., 1999) . To determine whether this was a characteristic specific to pulmonary artery endothelial cells, the growth characteristics of a human pulmonary microvascular endothelial cell type (HMVEC-L) and the widely used human umbilical vein endothelial cells (HUVEC) on 3-microm microporous filters were examined by transmission electron microscopy (TEM) . Similar to HPAEC, HMVEC-L and HUVEC were also found to grow on both sides of the filter . All three endothelial cell types were capable of migrating through the 3 microm pores of the filter to form a monolayer on the filter underside . The endothelial cells on the underside were orientated in an inverted position with the luminal surface facing away from the filter . Such 'bilayer' formation was observed at a range of seeding densities and in different culture media . Despite the presence of a bilayer of endothelial cells, TEM demonstrated that neutrophils migrated successfully across the cell-filter-cell system . Previous transmigration reports in which an in vitro model similar to ours was used have often assumed only one layer of endothelial cells . The observations reported here indicate that while endothelial cells on microporous filters are useful models for examining leukocyte-endothelial interactions, they are not appropriate for studies examining endothelial cell 'sidedness.'

J Periodontol, 1999 Aug, 70(8), 902 - 8
Involvement of cyclooxygenase-2 in interleukin-1alpha-induced prostaglandin production by human periodontal ligament cells; Noguchi K et al.; BACKGROUND: Human periodontal ligament (PDL) cells produce prostaglandin (PG) E2 in response to proinflammatory cytokines . However, the mechanism of PGE2 production is not well understood . The purpose of the present study was to investigate the involvement of cyclooxygenase (COX)-1 and COX-2 in PGE2 production by PDL cells stimulated with a proinflammatory cytokine, interleukin-1alpha (IL-1alpha), and to examine the regulation of PGE2 production by cell-cell interaction of human gingival keratinocytes and PDL cells . METHODS: The levels of PGE2 in the culture media of PDL cells stimulated with IL-1alpha or culture media of human gingival keratinocytes were determined by an enzyme-linked immunosorbent assay . Expression of COX-1 and -2 mRNA and protein was studied by Northern blot analysis and Western blot analysis, respectively . RESULTS: IL-1alpha-stimulated PDL cells produced PGE2 in a time-dependent manner . Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a selective COX-2 inhibitor, completely inhibited PGE2 production by the IL-1alpha-stimulated cells . COX-2 mRNA was detected after IL-1alpha stimulation, although it was not detected in unstimulated cells . There was no difference in expression of COX-1 mRNA between unstimulated cells and IL-la-stimulated cells . Expression of COX-2 protein in IL-1alpha-stimulated cells was increased, compared with that in unstimulated cells, whereas COX-1 protein expression was almost the same in both the cells . Treatment of IL-1alpha-stimulated PDL cells with dexamethasone, known to inhibit COX-2 expression, prevented PGE2 production and COX-2 mRNA expression . Addition of the culture media of human gingival keratinocytes to PDL cells increased PGE2 production . The PGE2 production was depressed by treatment of the cells with IL-1 receptor antagonist and anti- IL-1alpha antibody, not with anti-IL-1beta antibody . The PGE2 production was also inhibited by treatment with NS-398 and dexamethasone . CONCLUSIONS: We suggest that PDL cells stimulated with IL-1alpha produce PGE2 through de novo synthesis of COX-2 and that the cell interaction of gingival keratinocytes and PDL cells controls COX-2 expression and PGE2 production via IL-1alpha or 1alpha IL-la-like factor(s) . Selective COX-2 inhibitors, which have the advantage of reduced gastric toxicity, may provide a useful approach to treatment of periodontal disease.

Invest Ophthalmol Vis Sci, 1999 Sep, 40(10), 2418 - 22
Synthesis and release of docosahexaenoic acid by the RPE cells of prcd-affected dogs; Chen H et al.; PURPOSE: Dogs affected with progressive rod-cone degeneration (prcd) have reduced levels of docosahexaenoic acid (DHA, 22:6n-3) in their plasma and rod photoreceptor outer segments (ROS) . Dietary supplementation of DHA has failed to increase the ROS DHA levels to that of unaffected control dogs . The present study was undertaken to test the hypothesis that prcd-affected dogs have a reduced capacity for the synthesis and/or release of DHA in retinal pigment epithelial (RPE) cells . METHODS: RPE cells (first passage cultures) from prcd-affected and normal dogs were incubated with {3H}eicosapentaenoic acid (EPA, 20:5n-3) for 24 and 72 hours . After incubation, the radiolabeled fatty acids in the cells and media were analyzed . RESULTS: DHA and all its metabolic intermediates were detected in RPE cells from prcd-affected and normal dogs . No significant difference was found in the amount of products (including DHA) synthesized between normal and affected RPE cells at either time point . In the culture media, RPE cells from prcd-affected dogs released significantly more DHA than cells from normal dogs after 72-hour incubation, but not after 24-hour incubation . CONCLUSIONS: RPE cells from prcd-affected dogs can synthesize and release DHA at least as efficiently as cells from normal dogs . Therefore, synthesis of DHA from its precursor and its release from RPE cells does not appear to contribute to the reduction in ROS DHA levels found in prcd-affected animals.

Am J Reprod Immunol, 1999 Aug, 42(2), 95 - 9
Progress in characterization of pre-implantation factor in embryo cultures and in vivo; Barnea ER et al.; PROBLEM: Pre-implantation factor (PIF), a small, embryo-derived peptide is detected in the maternal serum prior to implantation and is associated with successful pregnancy outcome . However, its identity is not known . METHOD OF STUDY: PIF was isolated from mouse embryo conditioned media and from pregnant porcine sera, using high-performance liquid chromatography (HPLC) followed by mass spectrometry . RESULTS: Conditioned culture media was separated by gel filtration chromatography followed by reversed phase chromatography . At each step, PIF activity was determined by the lymphocyte/platelet binding autorosette assay (LPBA) . Mass spectrometry yielded a single peak with a mass of 1300 Da . The peptide is, however, present in very low concentrations (fM), which has so far precluded complete identification . Pregnant porcine sera that exhibit potent PIF activity were deproteinated by acetone and further fractionated by reversed phase HPLC . Active fractions contain peptides of molecular masses 523 and 551 Da . CONCLUSION: PIF, likely to be peptides, represents a novel substance related to pregnancy initiation and maintenance.

Biorheology, 1998 Jul-Oct, 35(4-5), 245 - 61
Shear stress gradient over endothelial cells in a curved microchannel system; Frame MD et al.; Our purpose was to test a scale model of the microcirculation by measuring the shear forces to which endothelial cells were exposed, and comparing this to computer simulations . In vitro experiments were performed to measure the 2-dimensional projected velocity profile along endothelial cell lined microchannels (D-shaped, 10-30 microns radius, n = 15), or in microchannels without endothelial cells (n = 18) . Microchannels were perfused with fluorescently labeled microspheres (0.5 micron dia., < 1%) suspended in cell culture media . The velocity of individual microspheres was obtained off-line (videorecording), using an interactive software program; velocity was determined as the distance traveled in one video field (1/60 s) . Mass balance was verified in the microchannels by comparing the microsphere velocities to the perfusion pump rate . In confluent endothelial cell lined microchannels, a velocity profile was obtained as microspheres passed an endothelial cell nucleus (identified by fluorescent dye), and again, for a paired region 100 microns away without nuclei (cytoplasm region) . The velocity profile was significantly shifted and sharpened by the endothelial cell nucleus, as anticipated . Over the nucleus, data are consistent with a normal sized nucleus extending into the lumen, further confirming that this scale model can be used to determine the wall shear stress to which endothelial cells are exposed . Using the experimental bulk phase fluid parameters as boundary conditions, we used computational fluid dynamics (CFD) to predict the expected wall shear stress gradient along an endothelial cell lined D-shaped tube . The wall shear stress gradient over the nucleus was 2-fold greater in the radial versus axial directions, and was sensitive to lateral versus midline positioned nuclei.

Ophthalmic Res, 1999, 31(6), 416 - 25
Fetal calf serum protects cultured porcine corneal endothelial cells from endotoxin-mediated cell damage; Sobottka Ventura AC et al.; In corneal organ culture, a contamination of sterile culture media with endotoxin is frequently found . Thus, we investigated if the presence of endotoxin affects the viability of cultured porcine corneal endothelial cells . Endotoxin in high concentrations caused morphological cell changes in porcine corneal endothelial monolayer cultures, delayed proliferation and decreased cellular esterase activity of porcine corneal endothelial cells in vitro . The toxic effect of endotoxin was modulated by the fetal calf serum content of the medium, the concentration of endotoxin and the incubation time.

Ophthalmic Res, 1999, 31(6), 399 - 406
The human fetal retinal pigment epithelium: A target tissue for thyroid hormones; Duncan KG et al.; Thyroid hormone (T(3)) has previously been shown to regulate visual function in experimental animals and humans . To determine if T(3) exerts direct effects on retinal function, cultured human fetal retinal pigment epithelial (RPE) cells were tested for the presence of thyroid hormone receptors (TRs) and T(3) responses . Using TR-isoform-specific reverse-transcriptase polymerase chain reaction techniques, mRNA was detected for alpha1, alpha2 and beta1 TR isoforms . Immunohistochemistry using a polyclonal antibody that simultaneously recognizes alpha1, alpha2 and beta1 TRs showed nuclear staining of the fetal RPE . Specific binding of (125)I-T(3) to RPE cell nuclear extracts was detected, and Scatchard analysis revealed a K(d) of 110 pM . To determine if RPE cells can respond to T(3), hyaluronic acid (HA) levels in cell culture media were measured after 2, 4 or 6 days of growth in medium containing 10(-7) M T(3) . T(3) inhibited accumulation of HA in the cell culture medium of RPE cells . This effect was not evident at 2 days, but at 4 days there was 42.8% less HA in cell culture medium of RPE cells grown in 10(-7) M T(3) (p < 0.01, t test) . The effect persisted through 6 days, when there was 46.3% less HA in cell culture medium of RPE cells grown in 10(-7) M T(3) (p < 0.001, t test) . The data indicate that human fetal RPE cells are a direct target for thyroid hormones.

Obstet Gynecol, 1999 Sep, 94(3), 435 - 40
Stromelysins in placental membranes and amniotic fluid with premature rupture of membranes; Fortunato SJ et al.; OBJECTIVE: To determine the expression and site of production of stromelysins in fetal membranes and to measure stromelysin 1 levels in amniotic fluid and amniochorion culture media . METHODS: Amniochorionic membranes were cultured from organ explant . Membranes were stimulated with lipopolysaccharide for 24 hours after a 48-hour preincubation period . Membranes were also collected from women after vaginal deliveries . RNA samples from those tissues were subjected to reverse transcriptase-polymerase chain reaction using primers specific for stromelysin 1, stromelysin 2, stromelysin 3, and matrilysin . In situ hybridization and immunohistochemistry were used to localize stromelysin mRNA and peptide . Levels of stromelysin 1 in culture media and amniotic fluid collected from women with preterm premature rupture of membranes (PROM) and at term with intact membranes were compared using enzyme-linked immunosorbant assay . RESULTS: Amniochorion in culture and from laboring and nonlaboring women expressed all three stromelysins . In situ hybridization showed stromelysin mRNA in amnion, chorion, and extracellular matrix . Immunohistochemical analysis localized stromelysin 1 protein to those same regions . Amniotic fluid levels of stromelysin 1 were higher in preterm PROM amniotic fluids (median 3.2 ng/mL) compared with term deliveries with intact membranes (median 1.3 ng/mL) (P = .02) . Lipopolysaccharide stimulation in culture increased the release of stromelysin 1 from fetal membranes compared with control (median 70.35 versus 15.8 ng/mL, respectively, P = .05) . CONCLUSION: Human fetal membranes are a source of stromelysins 1, 2, and 3 . Increased stromelysin 1 during preterm PROM and in vitro after lipopolysaccharide stimulation suggests a possible effect of that matrix metalloproteinase in PROM.

J Invest Dermatol, 1999 Sep, 113(3), 398 - 402
Aberrant cutaneous expression of the angiogenic factor midkine is associated with neurofibromatosis type-1; Mashour GA et al.; Neurofibromatosis type 1 is a common autosomal dominant disorder (incidence 1:3500) characterized by lesions that include neural crest derivatives such as Schwann cells and melanocytes . A critical event in the pathogenesis of neurofibromatosis type 1 is the heterozygous germ-line loss of the tumor suppressor gene NF1 . Additional genetic and/or epigenetic events have been posited, including various alterations in growth factor expression . By in situ hybridization and immunohistochemistry, we demonstrate aberrant expression of the angiogenic and tumorigenic growth factor midkine in the skin of patients with neurofibromatosis type 1, but not normal individuals . We demonstrate that midkine expression is independent of the presence of neurofibromas, and thus appears to be associated with mutations in the NF1 gene . Furthermore, midkine-containing culture media is shown to stimulate the growth of human endothelial and neurofibroma-derived cells . In conclusion, we introduce the skin as a source of dysregulated growth factors in neurofibromatosis type 1, and suggest the further study of the angiogenic factor midkine in neurofibromatosis type 1 pathogenesis.

Histochem J, 1999 Jun, 31(6), 403 - 9
Effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of decorin and biglycan by cultured osteoblastic cells; Takagi M et al.; The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta (TGF-beta) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2) . Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment . These results were evident 6 days after treatment . However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells . Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24 h with graded concentrations of TGF-beta1 (0, 0.1, 1.0, 5.0, and 10 ng/ml) . TGF-beta1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression . Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-beta1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells . These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA . Both rhBMP-2 and TGF-beta1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.

In Vitro Cell Dev Biol Anim, 1999 Jul-Aug, 35(7), 394 - 402
Quantitative aspects of the selective killing of transformed cells by methotrexate in the presence of leucovorin; Chow M et al.; A quantitative study was made of the cytotoxicity of methotrexate (MTX) for nontransformed and transformed NIH 3T3 cells in the presence and absence of leucovorin . The study was preceded by an analysis of the growth rates of the cells at low and high population density combined with low and high concentrations of calf serum (CS) . The reduced maximal growth rates of the transformed cells at low population densities relative to the nontransformed cells reinforced earlier evidence that heritable damage involving chromosome aberrations drives the process of transformation . When small numbers of transformed cells are cocultured with a large excess of nontransformed cells in the assay for transformed foci, the transformed cells were more readily killed by MTX than the nontransformed cells . The selectivity was increased when leucovorin (folinic acid) was present in the medium . The selective killing of the transformed cells actively multiplying in foci was most pronounced when the background of nontransformed cells had become confluent and their growth was inhibited . However, selectivity has also been demonstrated when transformed and nontransformed cells are growing at their maximum rates at low density despite the lower growth rate of the transformed cells under these conditions . The sensitivity of transformed cells in pure culture to MTX was lower during the first 3 d of subculture than in the following 6 d but decreased to zero a few d after net growth had ceased . The nontransformed cells were more susceptible to killing by MTX in Dulbecco's modified Eagle's medium (DMEM) than in MCDB 402, but the transformed cells were sensitive to MTX in both media . The high selectivity of MTX for transformed over nontransformed cells in MCDB 402 results from the presence of 1.0 microM leucovorin (5-formyltetrahydrofolate), a reduced form of the folic acid present in most other culture media . When leucovorin was added to DMEM with its high concentration of folic acid, the resistance to MTX of both nontransformed and transformed cells was greatly increased, but the selectivity of MTX for transformed cells was almost entirely lost . The results indicate that leucovorin protects nontransformed cells against concentrations of MTX that kill transformed cells, but the protection is dependent on the relative amounts of leucovorin to folic acid in the medium . The relative sensitivities of transformed and nontransformed cells in our system to MTX when both cell types are exhibiting their characteristic differential in growth behavior is similar to that described for tumor and normal cells in vivo . Since the unregulated growth behavior of the transformed, tumor-producing cells is efficiently and quantitatively measured in this system, it can be used to develop general principles of treatment and resolve questions of cytotoxic mechanism.

J Parasitol, 1999 Aug, 85(4), 663 - 71
Immunological characterization of antigens released by Trypanosoma cruzi-infected cells; Grijalva MJ et al.; Chagas' disease, caused by Trypanosorna cruzi, is characterized by the appearance of pathological lesions in the heart and other tissues during the chronic phase . The mechanisms responsible for such damage are still unclear . In the vertebrate host, T . cruzi replicates intracellularly before transforming from amastigotes into trypomastigotes . The infected host cell then lyses, releasing the cytoplasmic contents and the parasites that shed membrane glycoproteins soon after release . The sum of all these components we have termed released antigen (Rag) . We characterized antigens, released in vitro by fibroblasts infected with T . cruzi, obtained by concentrating conditioned serum-free culture media . The results demonstrate that Rag contains a complex protein mixture including stage-specific T . cruzi antigens (Ssp-1, -2, -4), glucose-regulated protein (Grp) 78h, and peptides recognized by the monoclonal antibody 2B10 . These peptides exhibit neuraminidase activity and are expressed by intracellular and 10-20% of released trypomastigotes . Additionally, Rag is recognized by sera from T . cruzi-infected mice and human chagasic patients . Rag also stimulates in vitro production of interferon-gamma by splenocytes from resistant C57B1/6 and susceptible BALB/c infected mice and interleukin-4 by splenocytes from BALB/c infected mice . Altogether these results indicate that Rag is immunologically relevant and could contribute to pathogenesis of T . cruzi infection.

Klin Lab Diagn, 1999 Jun, (6), 35 - 7
{Activation of lymphocyte nucleoli in culture adding the putative allergen to culture media: a tool for allergy diagnosis}; Bykova IA et al.; A method for diagnosis of allergy from activation of the peripheral blood lymphocyte nucleoli in short-lived cultures of peripheral blood leukocytes of 16 patients with clinical manifestations of drug allergy is described . The tests were positive in 75% cases.

Biotechnol Bioeng, 1999 Oct 20, 65(2), 192 - 200
Cell lines with reduced UDP-N-acetylhexosamine pool in the presence of ammonium; Cayli A et al.; The glycosylation of pharmaglycoproteins from recombinant cell lines can be affected by an uncontrolled accumulation of ammonium in the medium . Glucosamine-6-phosphate isomerase (GPI) has been proposed as the key enzyme responsible for elevating the intracellular UDP-N-acetylhexosamine pool (UDPGNAc) by accepting ammonium from the medium of cultured mammalian cells . As previously reported, the increased UDPGNAc pool then affects the N-glycan complexity in glycoproteins . To understand the entry of extracellular ammonium into the cellular metabolism, GPI has been isolated to homogeneity from BHK-21 cells and characterized . Thus, the complete pathway by which ammonium enters the cellular metabolism was elucidated . To reduce the negative effects of ammonium, GPI was inhibited using two different strategies . First, the addition of mannose to the culture media and, second, antisense RNA expression . In both cases, the cellular UDPGNAc pool was suppressed in the presence of high ammonium concentrations in the medium . However, constant suppression of the UDPGNAc pool could not be achieved by antisense RNA expression because antisense clones were apparently unstable . Further studies showed that the main reason for instability was the inducibility of GPI by its substrate ammonium . GPI was induced to a factor of two under ammonium-containing medium conditions . We propose gene knockout technology for GPI repression to obtain cell lines consisting of an UDPGNAc pool unaffected by the presence of ammonium .

Bone, 1999 Aug, 25(2), 205 - 9
Cloning and expression of rhesus monkey cathepsin K; Guay J et al.; Cathepsin K is a cysteine protease involved in degradation of human type I collagen and plays a primary role in bone resorption . We have cloned rhesus monkey cathepsin K by reverse transcriptase-polymerase chain reaction (RT-PCR) from rhesus ovary poly A+ RNA . The sequence for the rhesus enzyme is 98% identical to that of the human with 100% identity within the mature active form of cathepsin K . Rhesus monkey cathepsin K was transiently expressed in Chinese hamster ovary (CHO) cells and found to be secreted as the proenzyme in the culture media and 50% activated to the mature form intracellularly . The substrate specificity preference of aminomethylcoumarin and rhodamine peptide substrates was Leu > Phe > Pro in the P2 position when tested with constant arginine at P1 . The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC50s of 3.5 nmol/L and 13 ng/mL, respectively . The apparent second order rate constants of inactivation by E-64 were 66,000 M(-1) s(-1) and 130,000 M(-1) s(-1) for the recombinantly expressed rhesus monkey and human cathepsin K, respectively . The high similarity between the sequences and the kinetic properties of rhesus monkey and human cathepsin K establishes this monkey species as a suitable animal model for development of novel cathepsin K inhibitors as antiresorptive agents.

J Biol Chem, 1999 Aug 27, 274(35), 25085 - 92
Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties; Itano N et al.; Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned . In this study, we characterized and compared the enzymatic properties of these three HAS proteins . Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat . The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants . Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA . Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da . Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da) . The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.

Pharmacology, 1999 Sep, 59(3), 135 - 41
Effects of ambroxol on spontaneous or stimulated generation of reactive oxygen species by bronchoalveolar lavage cells harvested from patients with or without chronic obstructive pulmonary diseases; Teramoto S et al.; We examined the effects of ambroxol on spontaneous or stimulated generation of reactive oxygen species (ROS) by bronchoalveolar lavage (BAL) cells prepared from 6 patients with chronic obstructive pulmonary disease (COPD) and age-matched control subjects without COPD . The ROS produced by BAL cells were measured by the lucigenin-dependent chemiluminescence method . The application of ambroxol into culture media containing BAL cells inhibited spontaneous and stimulated generation of ROS by BAL cells harvested from COPD patients and control subjects in an ambroxol concentration-dependent manner . These results indicate that ambroxol may be a candidate agent for reducing oxidant stresses of airways in COPD.

Exp Nephrol, 1999 Jul-Aug, 7(4), 306 - 13
Transdifferentiation of distal but not proximal tubular epithelial cells from human kidney in culture; Baer PC et al.; Human renal proximal and distal (thick ascending limb and early distal convoluted tubule) epithelial cells have been isolated according to their specific antigen expression . The cells were well characterized by flow cytometry, enzyme cytochemistry and electron microscopy and cultured for up to 3 months . Cultured tubular cells coexpressed cytokeratin and vimentin as intermediate filament proteins . While primary isolated cells, proximal as well as distal, revealed the phenotypic characteristics of their nephron origin, cultured distal cells showed the tendency to dedifferentiate/transdifferentiate . Distal cells lost their characteristic expression of Tamm-Horsfall glycoprotein and started de novo expression of the proximal marker proteins aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV . The expression of these antigens by distal cells could be shown by flow-cytometric analysis and fluorescence microscopy . Enzyme activity assays revealed the activity of aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV, but not of the proximal marker enzyme alkaline phosphatase . This antigenic shift could not be prevented in different culture media, and the original phenotype could not be restored . Cultured cells displayed characteristic hormonal stimulation patterns indicative of their proximal and distal origins, as shown by activation of adenylate cyclase by different peptide hormones . These results indicate that distal tubular cells possibly transdifferentiate to a more proximal phenotype in view of loss of the distal marker enzyme Tamm-Horsfall protein and de novo expression of proximal marker enzymes like dipeptidyl peptidase IV and aminopeptidase M.

J Biomed Mater Res, 1999 Nov, 47(2), 139 - 51
Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)(2)D(3) through regulation of phospholipase A(2) activity and activation of protein kinase A; Lohmann CH et al.; Implant surface roughness influences osteoblast proliferation, differentiation, and local factor production . Moreover, the responsiveness of osteoblasts to systemic hormones such as 1, 25-(OH)(2)D(3) is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1, 25-(OH)(2)D(3) are synergistic . Prostaglandin E(2) (PGE(2)) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)(2)D(3) . However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1, 25-(OH)(2)D(3) . The present study examined the potential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), and protein kinase C (PKC) in this process . MG63 osteoblast-like human osteosarcoma cells were cultured on cpTi disks with R(a) values of 0 . 54 microm (PT), 4.14 microm (SLA), or 4.92 microm (TPS) . PKA was inhibited by adding H8 to the cultures; similarly, PLA(2) was inhibited with quinacrine or activated with melittin, and PKC was inhibited with chelerythrine . Inhibitors or activators were included in the culture media through the entire culture period or for the last 24 h of culture . In addition, cultures were treated for 24 h with inhibitors or activators in the presence of 1,25-(OH)(2)D(3) . The effects on cell number and alkaline phosphatase specific activity were determined after 24 h; PKC activity was determined after 9 min and at 24 h . Cell number was reduced on rough surfaces, and alkaline phosphatase activity was increased . 1,25-(OH)(2)D(3) had a synergistic effect with surface roughness on alkaline phosphatase . However, neither surface roughness nor 1,25-(OH)(2)D(3) had an effect on PKC . H8 treatment for 24 h inhibited cell number and alkaline phosphatase on all surfaces; however, when it was present throughout the culture period, the PKA inhibitor had no effect on cell number, but decreased alkaline phosphatase-specific activity . H8 reduced the 1,25-(OH)(2)D(3)-mediated effect on cell number and alkaline phosphatase . Quinacrine inhibited cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25-(OH)(2)D(3)-dependent decreases in both parameters . Melittin had no effect when applied for 24 h and did not modify the 1,25-(OH)(2)D(3) effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity . Chelerythrine, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period . However, it decreased alkaline phosphatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period . The results indicate that surface roughness and 1,25-(OH)(2)D(3) both mediate their effects through PLA(2) which catalyzes the rate-limiting step in PGE(2) production . Further downstream, PGE(2) activates PKA . Surface roughness-dependent effects are also mediated through PKC, but only after the cells have reached confluence and are undergoing phenotypic maturation . The effect of surface roughness on responsiveness to 1,25-(OH)(2)D(3) is mediated through PLA(2)/PKA and not through PKC .

Biochem Pharmacol, 1999 Sep 1, 58(5), 773 - 8
Inhibitory effect of nitric oxide on chemically induced differentiation of human leukemic K562 cells; Chenais B et al.; The effect of nitric oxide (NO) was investigated in the human K562 cell line during chemically induced erythroid differentiation . Butyric acid (BA) and the anthracycline antitumour drugs aclarubicin (ACLA) and doxorubicin (DOX) were used as differentiating agents . In all cases, cell hemoglobinization was dose dependently inhibited by NO donors such as sodium nitroprusside (SNP) . A 50% inhibition of cell differentiation was obtained with 25 microM SNP, which generated less than 2 microM nitrite in 3-day culture media . Increasing SNP concentrations led to higher nitrite accumulation (up to 12 microM with 1 mM SNP) and total inhibition of cell hemoglobinization, but did not have a significant effect on cell proliferation . As shown by Northern blotting, high concentrations of SNP (1 mM) reduced the expression of gamma-globin and porphobilinogen deaminase, but did not change GATA-1 and NF-E2 mRNA levels in ACLA- and BA-treated cells . In contrast, hemin-induced erythroid differentiation was not affected by the presence of NO donors . Altogether, these results show that NO is able to inhibit cell differentiation induced by some (ACLA, DOX, BA), but not all (hemin), agents . The inhibitory effect of NO seems to take place downstream of the regulation of erythroid gene expression.

Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 76 - 9
Adrenergic stimulation of hepatocyte growth factor expression; Broten J et al.; Hepatocyte growth factor (HGF), a potent mitogen, is released into plasma at increased levels following injury to certain tissues, including the liver . Early increases in plasma HGF are not due to a release from the injured liver, but rather from distal organs, particularly the lung . We have investigated the ability of norepinephrine (NE), which rises rapidly in plasma after liver resection, to trigger elevated production of HGF in MRC-5 human embryonic lung fibroblasts . Levels of HGF released to culture media and of HGF mRNA increased when cultures were exposed to NE, or to other adrenergic agonists . While stimulation of either beta- or alpha(1)-adrenergic receptors increased HGF expression, responses to NE appear to be mediated primarily via beta receptors . Since NE has already been shown to act as a comitogen with HGF, our findings suggest that adrenergic hormones may act both to induce production of HGF at distal sites, and to enhance the response to HGF at target tissues .

Eur J Biochem, 1999 Aug, 264(1), 49 - 54
Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides; Lin Q et al.; Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs) . The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-' . In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs . The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity . Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs . However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs . These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3510 - 7
A novel potent strategy for gene delivery using a single peptide vector as a carrier; Morris MC et al.; We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides into cultured cells . Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells . In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects them from degradation in cell culture media . MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids into several different cell lines in only 1 h . Moreover, MPG enables complete expression of the gene products encoded by the plasmids it delivers into cultured cells . Finally, we have investigated the potential of MPG as an efficient delivery agent for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene . MPG efficiently delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C expression levels and promoted a block to cell cycle progression . Based on our results, we conclude that MPG is a potent delivery agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its contribution to the development of new gene therapy strategies could be of prime interest.

J Clin Endocrinol Metab, 1999 Aug, 84(8), 2978 - 81
Effects of TGF-beta1 on proliferation and IGFBP-3 production in a primary culture of human fetal epiphyseal chondrocytes (HFEC); Garcia-Ramirez M et al.; Proliferation and differentiation of chondrocytes from growth cartilage are modulated by hormones and growth factors, among which TGF-betas have been recognized as some of the more potent regulators although their specific cell effects on cartilage physiology are not fully understood . Primary human fetal epiphyseal chondrocytes (HEFC) constitutively produce TGF-beta1 at different times of culture progression . Treatment of 48-h . serum-deprived semiconfluent HFEC with 0.1-50 ng/ml of TGF-beta1 for 48-h . decreased (3H)Thymidine incorporation by 25-50 % and cell number by 25 % . In addition, IGFBP-3, the main insulin-like bonding protein produced by HFEC, showed a slight increase by TGF-beta1 in culture media . The changes in IGFBP-3 protein levels correlated well with its mRNA, indicating that TGF-beta1 is able to up-regulate IGFBP-3 synthesis in chondrocytes . Nevertheless, the IGFBP-3 accumulation in culture media does not produce a clear growth inhibitory effect on chondrocytes . Thus, we conclude that even though TGF-beta1 is able to up-regulate IGFBP-3, the growth inhibitory action produced by TGF-beta1 is not mediated by IGFBP-3 increase and appears to be mainly a direct TGF-beta1 effect on HFEC.

Acta Histochem, 1999 Jul, 101(3), 305 - 16
Expression of multiple matrix metalloproteinases and urokinase type plasminogen activator in cultured Kaposi sarcoma cells; Meade-Tollin LC et al.; Kaposi's sarcoma (KS) cells are considered to be of endothelial origin . KS lesions are characterized by hyperproliferation and an invasive phenotype . We have determined that KS cell cultures constitutively secrete multiple forms of several matrix metalloproteinases (MMPs) and an altered form of urokinase plasminogen activator (uPA) by zymogram and Western analysis of the culture media . MMPs are a family of secreted endoproteinases which degrade components of the extracellular matrix . Their enhanced expression and activity are strongly correlated with cellular processes involving tissue remodeling and invasion . The KS cells secrete increased levels of gelatinase A and B and a high molecular weight uPA in vitro when compared with non-KS endothelial or epithelial cells . Multiple forms of gelatinases A and B were observed on gelatin zymograms . Caseinolytic bands observed were confirmed by Western blot analysis to be due to stromelysin activity, whereas matrilysin was not detected by casein zymography . Western blot analysis also detected secretion of interstitial collagenase and high molecular weight uPA . Gelatinolytic activity with the mobility of gelatinase B was detected on gelatin zymograms, but not by Western analysis . This unusual constitutive expression pattern of MMPs and uPA by KS cells in vitro is characterized by elevated levels of gelatinase A, gelatinase B, interstitial collagenase, stromelysin and a high molecular weight form of uPA, and the lack of expression of matrilysin . These secreted MMPs, taken together, are capable of digesting a broad range of components of the extracellular matrix . This unusual pattern is likely to contribute to the characteristic hyperproliferative and invasive phenotype of KS lesions.

Ophthalmology, 1999 Aug, 106(8), 1498 - 9
Live virus survives excimer laser ablation; Taravella MJ et al.; OBJECTIVES: To determine whether live virus can withstand excimer laser ablation and pose a possible health hazard to medical personnel . DESIGN: Experimental study . METHODS: Fibroblasts infected with oral polio vaccine virus were ablated with an excimer laser . The plume was collected using a smoke evacuator and bubbled through viral culture media . MAIN OUTCOME MEASURES: The inlet tube from the smoke evacuator was swabbed and cultured for virus . Liquid from the bubble trap was also cultured . RESULTS: Live virus was shown in the material trapped from the laser plume . CONCLUSIONS: Oral polio vaccine virus can survive excimer laser ablation . Whether other more clinically relevant viruses, such as human immunodeficiency virus, can withstand ablation and remain infectious remains to be determined.

J Virol, 1999 Sep, 73(9), 7210 - 7
An endoplasmic reticulum retrieval signal partitions human foamy virus maturation to intracytoplasmic membranes; Goepfert PA et al.; Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes . The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER . This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif . Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses . Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses . Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers . We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.

Vet Immunol Immunopathol, 1999 May, 68(2-4), 177 - 86
Beta-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes; Suriyasathaporn W et al.; The role of ketone bodies on chemotactic capacities of leukocytes was characterized in two experiments . Experiment I was performed to investigate the association between serum beta-hydroxybutyrate concentrations (BHB) and in vitro chemotaxis of leukocytes . Cows were divided into low-BHB, medium-BHB, and high-BHB ones and classified according to their BHB . Leukocytes from high-BHB cows had a significantly lower chemotactic differential than leukocytes from low-BHB cows (p < 0.01) . The effect of adding ketone bodies into in vitro chemotaxis cultures on leukocytes chemotaxis was studied in Experiment II . Either individual or a combination of commercial ketone bodies - sodium salts of BHB (BHBA), lithium salt of acetoacetate (ACAC), and acetone (Acetone) - were diluted in culture media and divided into eight concentrations corresponding to concentrations of bovine subclinical and clinical ketosis . For leukocytes from medium- and high-BHB cow, the chemotactic indexes of leukocytes were reduced by ACAC and Acetone . Chemotactic differentials of cultures with ACAC and acetone supplementation from both sources of leukocytes were significantly lower than that of the control culture (p < 0.05) . For leukocytes from high-BHB cows, chemotactic indexes were suppressed in a ketone-body environment . In conclusion, leukocytes from naturally-occurring ketotic cows have lower chemotactic differentials than those from non-ketotic cows, and a chemotactic capacity indicated by a chemotactic differential is impaired when leukocytes migrate in an environment with ketone bodies in vitro.

Bull Acad Natl Med, 1999, 183(4), 815 - 22; discussion 822-4
{Dynamic studies of Langerhans cell histiocytosis cells . Their contribution to the current concept of the disease . Report of a series of 38 cases}; Nezelof C et al.; In vitro explantation of 38 fragments of eosinophilic granuloma of bones was attempted . A satisfactory growth was obtained in nearly 90% of cases . This short-term culture maintained the ultrastructural characteristics and, to a lesser extent, the cytochemical features of the Langerhans cells, confirming the Langerhans cell origin of this cell proliferation . In addition, this procedure was able to demonstrate an immunodependent erythrophagocytosis (3/3) and a preferential fixation of labelled precursors (Glycerol, choline of lipid metabolism as well as labelled dopamine (2/2) . All attempts to obtain a permanent cell line and graft to nude mice (even irradiated) failed . Under the in vitro conditions, the Langerhans cells do not divide up but can be readily identified up to 2 or 3 weeks . The contrast between the evident in vivo proliferation and the in vitro quiescent state suggests that some undetermined growth-factors targeted to the Langerhans cell system are missing in our commonly used culture media . The in vitro culture procedure could be of some help to their identification.

Int J Clin Lab Res, 1999, 29(2), 80 - 4
Effects of sodium selenite on in vitro interactions between platelets and endothelial cells; Ricetti MM et al.; Selenium is an essential component of glutathione peroxidase enzymes, which protect cells against peroxidation and control concentrations of intracellular proxides . Since selenium deficiency is associated with an increased incidence of arterial thrombosis, we studied the effect of selenium on in vitro interactions between platelets and endothelial cells . Platelets from normal volunteers on a diet with (PLTSe+) or without (PLTSe-) selenium supplementation and human umbilical vein endothelial cells cultured in medium alone (ECSe-) or supplemented with Se (ECSe+) were used . The effect of in vivo administration or in vitro supplementation of selenium on platelet function was investigated in an aggregometry model designed for studying the interactions between platelets and endothelial cells using ADP and arachidonic acid as agonists . We observed that: (1) selenium-dependent glutathione peroxidase enzyme activity increased in both PLTSe+ and ECSe+, being about fivefold higher in the former; (2) platelet aggregation was inhibited by Se+ cells; (3) Se+ cells released less thromboxane B(2) (PLTSe+) and more 6-keto-prostaglandin F(1alpha) (ECSe+) than Se- cells; (4) when ECSe+ were treated with acetylsalicylic acid, the inhibitory effect of selenium on platelet aggregation disappeared; (5) the concentration of nitric oxide metabolites in Se+ culture media did not differ from that in Se- media . We suggest that an antithrombotic effect on the interactions between platelets and endothelial cells can be induced by stimulating glutathione peroxidase enzymes with selenium via a mechanism that is blocked by acetylsalicylic acid and is apparently unrelated to the biosynthesis of nitric oxide metabolites.

J Reprod Fertil, 1999 Mar, 115(2), 201 - 13
Identification and characterization of proteins synthesized de novo and secreted by the reproductive tract of the American alligator, Alligator mississippiensis; Buhi WC et al.; The objectives of this study were to identify, characterize and examine differences in proteins synthesized de novo and secreted by different regions of the reproductive tract of the American alligator, Alligator mississippiensis, during three reproductive (vitellogenic, gravid, post-clutch) and one non-reproductive state . After capture, alligators from lakes in north central Florida were anaesthetized, the reproductive tract excised aseptically, the size of any follicle determined, and different functional regions of the tract dissected out and partitioned for explant culture . Analysis of the biosynthetic activity indicated regional variations within the tract, differences among reproductive groups and region by status interactions . When oviductal regions were considered regardless of reproductive status, the greatest incorporation of {3H}Leu into secreted nondialysable macromolecules was by the anterior and posterior infundibulum and oviductal tube compared with the transition zone and the uterus . When status was included, the biosynthetic activity of the anterior and posterior portion of the tract in non-reproductive alligators was not different, whereas that of the posterior region of the reproductive group (vitellogenic, gravid, post-clutch) was significantly lower than that of the anterior region . This finding indicates that regulation of protein synthesis and secretion by the non-reproductive alligator tract is different from that in the tract of the reproductive group . Explant-conditioned media were analysed by one-dimensional and two-dimensional SDS-PAGE and fluorography . Sixteen major proteins in culture media were identified as de novo synthesized, by relative molecular weight, by isoelectric point and by differences in distribution determined for reproductive status and oviductal region . Six proteins were examined by N-terminal amino acid microsequence analysis . On the basis of a 29 amino acid sequence, the major oviductal protein, alligator protein 1 (aP1: M(r) 55,000, basic), found in the infundibulum and tube of vitellogenic alligators, was identical to the major protein isolated from alligator egg albumen . Four proteins (aP4-aP7) were sequenced and shown to be significantly related to immunoglobulin heavy chains from several species . This study demonstrated that a large number of proteins are synthesized de novo and released by the female alligator reproductive tract and that there are biosynthetic activity differences by reproductive status and region . Six proteins have been identified, several of which may be incorporated into alligator egg albumen and some of which appear to be different from proteins found in the egg albumens of other species.

Contracept Fertil Sex, 1999 Jun, 27(6), 440 - 8
{Oocyte of domestic mammals: a model for the study of in vitro maturation}; Mermillod P et al.; Oocyte maturation represents the final step of a long differentiation process that allows this very special cell to fully express its reproductive task . During maturation the oocyte nucleus, blocked at the late prophase of meiosis from the end of foetal life, resumes meiosis and progress to the metaphase II stage . Beyond these nuclear aspects, oocyte maturation also involves cytoplasmic modifications including well known morphological progression as well as poorly understood biochemical changes that are determinant for successful fertilisation and early embryo development . In physiological conditions, maturation occurs in the preovulatory follicle after the ovulatory surge of gonadotropins, in a complex and changing environment . This complexity leaded to the formulation of the first in vitro maturation systems involving tissue culture media supplemented with biological fluids . A more precise study of the effect of individual medium components allowed the design of more simple maturation conditions providing more reproducible results with less sanitary risks . Amongst maturation activating factors, the epidermal growth factor (EGF) seems to play a key function in several species . Other factors such as hormones, ovarian peptides (inhibin, activin) and other growth factors may also be involved but the interplay between these factors remains to be clearly established . Improvement of in vitro maturation techniques allowed to evaluate the importance of oocyte intrafollicular differentiation before maturation on the resulting developmental competence . The increased knowledge of the regulation of intrafollicular meiotic arrest now allow the design of a prematuration step to allow oocytes from smaller follicles to complete their differentiation in vitro . This improvement will allow a larger use of the huge reproductive potential stored in the ovary.

Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 1999 Aug, 16(4), 205 - 7
{Preliminary studies on gene therapy for lysosomal alpha-mannosidosis}; Sun H et al.; OBJECTIVE: Exploration of the feasibility of treating human lysosomal alpha-mannosidosis by gene therapy . METHODS: Retroviral vector-mediated transfer of human lysosomal alpha-mannosidase cDNA into the diseased cat skin fibroblasts . Detection of the enzymatic activity in the cells and their culture media at different pH conditions . Localization of the recombinant enzyme in the cells by chemical staining . Cross-correction of untransduced cat cells by incubation in medium containing the secreted enzyme in the absence and presence of mannose-6-phosphate or fetal calf serum . In vivo expression of the recombinant enzyme in organoids containing the vector-transduced cells . RESULTS: Among the two human cDNAs tested, only one encoded high levels of alpha-mannosidase activity in the cat cells, which shared the same pH profile and lysosomal localization with the normal enzyme . The recombinant enzyme was proportionally secreted into the medium of the cell culture and taken up by untransduced cells via mannose-6-phosphate receptor-mediated endocytosis . The uptake was partially inhibited in the presence of fetal calf serum, which was dose-dependent . In vivo, organoids containing the vector-transduced cells expressed detectable alpha-mannosidase activity for up to 6 weeks . CONCLUSION: The human cDNA was functional in the cat cells and the expression system could be used for development of gene therapy for lysosomal alpha-mannosidosis.

Fertil Steril, 1999 Jul, 72(1), 154 - 7
Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay; Ermilov A et al.; OBJECTIVE: To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition . DESIGN: In vitro study . SETTING: Academic medical center . PATIENT(S): None . INTERVENTION(S): None . MAIN OUTCOME MEASURE(S): Plasmid relaxation . RESULT(S): Ham's F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage . Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media . Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage . CONCLUSION(S): In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.

Carcinogenesis, 1999 Aug, 20(8), 1445 - 51
Matrix metalloproteinase inhibition prevents colon cancer peritoneal carcinomatosis development and prolongs survival in rats; Aparicio T et al.; Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation which play a role in cancer progression and metastatic spreading . We investigated the effects of the MMP inhibitor, batimastat, in vitro on the proliferation and invasiveness of the rat colon cancer cell line DHD/K12, and in vivo on the growth of an aggressive model of peritoneal carcinomatosis producing haemorrhagic ascites and metastases, obtained in the rat by i.p . injection of DHD/K12 cells . MMP production was studied in conditioned culture media, solid tumors and ascitic fluid . In vivo, after injection of tumor cells on day 0, rats received i.p . daily either batimastat (30 mg/kg) or equal volume of vehicle from day 2 until killing on day 43 (series I) or from day 13 until death (series II) . The grade of peritoneal carcinomatosis, ascite volume, number and size of liver metastases were evaluated in both series, and survival in series II . MMPs-1, -2 and -9 were identified in culture media, tumors and ascites . In vitro, batimastat did not modify DHD/K12 cell proliferation and slightly reduced cell invasion . In vivo, in series I, batimastat treatment totally prevented peritoneal carcinomatosis and liver metastasis development . In series II, it significantly prolonged survival (P < 0.0002) and reduced peritoneal carcinomatosis (P < 0.001) and hepatic metastases number as compared with controls . However, batimastat-treated rats of the two series had peritoneal inflammation with marked ascites . Nevertheless, inhibition of MMP is a new therapeutic approach which may be promising in treatment of microtumors as in more advanced cancer stages.

Nippon Ishinkin Gakkai Zasshi, 1999, 40(3), 169 - 73
{Biological characteristics of Emericella nidulans isolated from horse guttural pouch mycosis}; Kosuge J et al.; Seven strains of Emericella nidulans were isolated from lesions in the guttural pouch of horses with mycosis of this pouch . All strains grew in a wide range of temperature, pH and in five kinds of culture media . The optimum temperature that supported their growth was 38 degrees C, and all seemed to prefer an acidic environment with a pH of 4.0 . Proliferative conidial formation was found to be induced by the aforementioned temperature and pH . Moreover, detection of beta-haemolysis and protease production suggested that these strains are biologically and biochemically active, which may imply that they have a potent pathogenicity of their own . Characteristics of two other strains of E . nidulans isolated from fomites were the same as those isolated from the infected horses . These findings suggest that E . nidulans is a potentially pathogenic to horses.

Eur J Clin Microbiol Infect Dis, 1999 May, 18(5), 346 - 51
Early identification of Mycobacterium tuberculosis and Mycobacterium avium using the polymerase chain reaction on samples positive by a rapid commercial culture system; Sion C et al.; A combination of two methods -- a rapid culture method {Mycobacteria Growth Indicator Tube (MGIT); Becton-Dickinson, USA} and a double polymerase chain reaction (PCR) assay -- was assessed for the detection and identification of Mycobacterium tuberculosis and Mycobacterium avium from clinical samples . The aim of the study was to evaluate the ability of the system to offer rapid and accurate diagnosis of mycobacterial infections . After decontamination, clinical samples (n = 554) were stained and cultured in parallel on solid media and in MGITs following standard procedures . The performance of the two culture systems was compared . Positive MGITs were tested for the presence of Mycobacterium tuberculosis and Mycobacterium avium by PCR of IS6110 (Mycobacterium tuberculosis) and the 16S rRNA gene (Mycobacterium avium) . A total of 41 mycobacteria -- 27 Mycobacterium tuberculosis isolates, eight Mycobacterium avium isolates, and six other species of mycobacteria -- were isolated by one or both culture media . The MGIT system recovered 36 (87.8%) mycobacteria and the solid media 33 (80.4%) . The mean time to detection by the two culture systems did not differ overall, but the mean time to detection of Mycobacterium avium from smear-positive specimens was shorter in MGITs than in solid media (5.25 days vs . 16.25 days, P < 0.05) . The double PCR assay performed on the 36 positive MGITs correctly identified all 24 Mycobacterium tuberculosis-positive MGITs and all six Mycobacterium avium-positive vials . Therefore, application of the PCR assay to positive MGITs may mean that Mycobacterium tuberculosis and Mycobacterium avium can be identified at an earlier stage than with current methods.

Curr Eye Res, 1999 Aug, 19(2), 95 - 105
Ultrastructural evidence of mucus in human conjunctival epithelial cultures; Diebold YC et al.; PURPOSE . To demonstrate by ultrastructural techniques that human conjunctival epithelium cells in vitro can produce mucin-like secretion . METHODS . Primary cultures of human conjunctival epithelial cells were grown in different culture media . Cultures were allowed to grow and were processed after 5 days and 1, 2, 3, 4, or 5 weeks for transmission and scanning electron microscopy, according to the method of Nichols et al . modified in our laboratory . RESULTS . Marked differences were seen between primary cultures grown with or without hydrocortisone . A thick tannic acid-stained layer was observed when hydrocortisone was present in the culture medium; however, that layer was virtually absent in cultures grown with hydrocortisone-free media . Scanning electron microscopy revealed a dense deposit showing a network-like structure . Moreover, the age of the cultures clearly influenced the thickness of the tannic acid-stained deposit, which thickened as the cultures aged . CONCLUSIONS . These results strongly suggest that the layer growing in the presence of hydrocortisone is mucus . The fact that this material became more abundant as the cultures aged indicates that mucus is actively produced and secreted by conjunctival epithelial cells in vitro . This study might contribute to the knowledge of mucus-deficient pathologies of the ocular surface.

J Biol Chem, 1999 Jul 30, 274(31), 21783 - 9
Antisense oligonucleotides with different backbones . Modification of splicing pathways and efficacy of uptake; Schmajuk G et al.; A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2'-O-methyl sugar-phosphate oligonucleotides . The assay is based on modification of the splicing pathway of human beta-globin pre-mRNA . In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier . The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2'-O-methyl-oligoribonucleotides and 6-9- and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively . The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2'-O-methyl-oligoribonucleotides in free uptake from the culture media . The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells . Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.

Clin Chem Lab Med, 1999 May, 37(5), 593 - 9
Evaluation of an automated immunoassay method for cytokine measurement using the Immulite Immunoassay system; Berthier F et al.; Cytokines are key mediators in cell regulation and communication . The concentration of these proteins can rapidly and importantly increase during severe clinical situations . However, current techniques are not adapted to stat measurement, thus making their clinical use limited . In this context, the commercialization of five new kits for cytokine measurement interleukin ((IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and IL-2R) on an automated immunoanalyzer, the Immulite, seems to be a new approach for the determination of these markers . We report here the evaluation of the performance of these tests . The technique is based on a solid phase (bead) two site chemiluminescent enzyme immunometric assay . The analysis is performed within 60 to 90 minutes and the calibration is stable for 15 days . The values of the between-run imprecision study were similar to those from the within-run study with coefficients of variation (CV) ranging from 2% (low values of IL-8) to 11.5% for intermediate concentrations of IL-6 (500 pg/ml) . CVs were usually around 5% . The accuracy was determined by a linearity study using standards (except for IL-2R) provided by the National Institute for Biological Standards and Control (NIBSC) . Slopes obtained during this study were close to 1 (r2 = 0.99), except for IL-6, for which the slope was 1.55 . TNF-alpha values were close to those expected . IL-1 results were about 20% higher . IL-6 values were over estimated above 100 pg/ml and under estimated below this value . IL-8 study seemed to be impaired by the poor stability of this molecule in the NIBSC preparation . Correlation study with standard laboratory techniques gave variable results: for IL-1 (n = 43) the slope was 0.77 (study carried out using cell culture media), for IL-6 (n = 54) the slope was 0.78, for IL-8 (n = 37) the slope was 1.64, for TNF-alpha (n = 40) the slope was 0.33 and the slope for IL-2R (n = 51) was 5.1 . For the last cytokine, the unit in Immulite assay was different from the one used in our comparison technique . Cross-calibration results were consistent with these data and show that the bias is probably linked to a calibration problem . The study demonstrated excellent practicality of the system, and good stability of the calibration curve (15 days) . However, the sample volume required (350 microl for the IL-6 and the TNF-alpha) could constitute a limitation for pediatric measurements.

Arzneimittelforschung, 1999 Jun, 49(6), 541 - 3
Effects of D-methionine-containing solution on tumor cell growth in vitro; Sasamura T et al.; The effects of a nutrition therapy with D-methionine (Met)-containing solution were investigated in cell cultures of the AH109A cell line . The growth of AH109A hepatoma cells in culture media with D-Met-supplemented medium, L-Met-supplemented medium (control) and Met-free medium was compared . The D-Met-supplemented medium inhibited the cell growth to an extent similar to that manifested in the Met-free medium . The total free amino acid concentrations in the control medium decreased by approximately 40% on day 6 post-culture . However, the free amino acid concentrations in D-Met-supplemented and Met-free media did not change . Furthermore, alanine, which was not added to RPMI-1640, was detected in the control medium on day 6 post-culture . These results suggest the possibility of application of D-Met-containing solution to cancer patients receiving total parenteral nutrition.

Gen Comp Endocrinol, 1999 Aug, 115(2), 200 - 9
Induction of ovulation of mature oocytes by the maturation-inducing steroid 17,20beta,21-trihydroxy-4-pregnen-3-one in the spotted seatrout; Pinter J et al.; Incubation of mature, hydrated, follicle-enclosed oocytes of the spotted seatrout, Cynoscion nebulosus, with the maturation-inducing steroid (MIS), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), for 9-12 h resulted in the appearance of ovulated oocytes in the culture media . The ovulation response was concentration-dependent and steroid-specific . The other teleost MIS, 17, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), was also a potent inducer of ovulation, whereas progesterone and 11-deoxycorticosterone did not stimulate ovulation above control levels and partially antagonized the action of 20beta-S . The agonist and antagonist activities of these steroids on ovulation are consistent with their relative binding affinities for the ovarian nuclear progestogen receptor previously characterized in this species . Both the RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide blocked MIS-induced ovulation . This suggests that induction of ovulation by the MIS is through a genomic mechanism of action, and potentially involves the previously characterized nuclear progestogen receptor . Gonadotropin (hCG)-induced ovulation was blocked by addition of the steroid synthesis inhibitor cyanoketone, which was overcome by the addition of 20beta-S, but not pregnenolone . Thus, the most likely mechanism of gonadotropin-induced ovulation is an increase in the synthesis of the MIS . It is concluded that the processes of final oocyte maturation and ovulation are both regulated by the MIS . Whereas final oocyte maturation is mediated by the 20beta-S membrane receptor (P . Thomas and S . Das, 1997, Biol . Reprod . 57, 999-1007), ovulation is regulated by a genomic mechanism and is potentially mediated by the previously characterized nuclear progestogen receptor .

Biofactors, 1999, 9(2-4), 171 - 7
Role of plasma membrane coenzyme Q on the regulation of apoptosis; Lopez-Lluch G et al.; Serum withdrawal is a model to study the mechanisms involved in the induction of apoptosis caused by mild oxidative stress . Apoptosis induced by growth factors removal was prevented by the external addition of antioxidants such as ascorbate, alpha-tocopherol and coenzyme Q (CoQ) . CoQ is a lipophilic antioxidant which prevents oxidative stress and participates in the regeneration of alpha-tocopherol and ascorbate in the plasma membrane . We have found an inverse relationship between CoQ content in plasma membrane and lipid peroxidation rates in leukaemic cells . CoQ10 addition to serum-free culture media prevented both lipid peroxidation and cell death . Also, CoQ10 addition decreased ceramide release after serum withdrawal by inhibition of magnesium-dependent plasma membrane neutral-sphingomyelinase . Moreover, CoQ10 addition partially blocked activation of CPP32/caspase-3 . These results suggest CoQ of the plasma membrane as a regulator of initiation phase of oxidative stress-mediated serum withdrawal-induced apoptosis.

Biol Reprod, 1999 Aug, 61(2), 482 - 92
Comparison of protein synthesis patterns in mouse cumulus cells and mural granulosa cells: effects of follicle-stimulating hormone and insulin on granulosa cell differentiation in vitro; Latham KE et al.; Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells . Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo . Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious . Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro . Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells . Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased . The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone . Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development . The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes.

J Immunol Methods, 1999 Jun 24, 226(1-2), 29 - 41
A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology; Roden MM et al.; A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release . Target cells were loaded with Calcein-AM and then co-incubated with effector cells . An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media . Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells . Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells . The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets . In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates . The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.

Gan To Kagaku Ryoho, 1999 May, 26(6), 849 - 56
{Tumor metastases and adhesion molecules carbohydrates and lectins}; Nemoto Y et al.; The expression of carbohydrate antigens has been shown by retrospective immunohistochemical analysis to correlate to the progression and metastases of human cancers . However, the mechanisms of these changes of carbohydrate expression and the role of carbohydrates in the malignant behavior of tumor cells are not well known . In this article, we introduce methods to experimentally modify carbohydrate expression in tumor cells and to assess the involvement of these carbohydrate antigens in the malignant behavior of tumor cells . Modifications of the biosynthesis of O- and N-linked carbohydrates, and glycolipids are achieved by treating cultured tumor cells with culture media containing Benzyl-alpha-GalNAc, swainsonine, or D-PDMP, respectively . Enzymatic digestion of cell surface carbohydrates with sialidase, endo-beta-galactosidase or other glycosidases can also be performed . These cells can be used for short term experiments such as adhesion assays . However, modified carbohydrates may be recovered during in vitro and in vivo assays . By transfection of glycosyltransferase cDNA, or selection of tumor cells by binding lectins or antibodies, stable carbohydrate variant cells can be obtained which are suitable for long term experiments such as the experimental formation of metastases in vivo . The biological function of tumor cell surface carbohydrates may be diverse . These molecules are thought to influence adhesion interaction between tumor cells and the endothelial cells of target organs . However, carbohydrate recognition molecules, or lectins, are expressed on a variety of cells in the vascular system and in the immune system . Therefore, it is essential to design appropriate experimental models to study the biological significance of carbohydrate-lectin interactions in cancer progression and metastatic dissemination . Adhesion assays of tumor cells to selectin-transfected CHO cells were performed . Taking molecules other than selectins into consideration, adhesion assays using frozen tissue sections were also performed.

Am J Physiol, 1999 Jul, 277(1 Pt 1), L142 - 9
Mechanical strain-induced posttranscriptional regulation of fibronectin production in fetal lung cells; Mourgeon E et al.; We have shown that intermittent mechanical strain, simulating fetal breathing movements, stimulated fetal rat lung cell proliferation . Because normal lung growth requires proper coordination between cell proliferation and extracellular matrix remodeling, we investigated the effect of strain on fibronectin metabolism . Organotypic cultures of fetal rat lung cells, subjected to intermittent strain, showed increased fibronectin content in the culture media . Fibronectin-degrading activity in media from strained cells was similar to that of static cultures . Northern analysis revealed that strain inhibited fibronectin mRNA accumulation seen during static culture . Synthesis of fibronectin, determined by metabolic labeling, was increased by strain despite lower mRNA levels or presence of actinomycin D . This increase was not mediated via a rapamycin-sensitive mechanism . Strain stimulated prelabeled fibronectin secretion even in the presence of cycloheximide . These results suggest that strain differentially regulates fibronectin production of fetal lung cells at the transcriptional and posttranscriptional levels . Mechanical strain increases soluble fibronectin content by stimulating its synthesis and secretion without increasing fibronectin message levels.

Dev Biol Stand, 1999, 99, 167 - 80
Safety issues of animal products used in serum-free media; Merten OW; The development of media free of serum and animal or human protein is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination . The main drawback associated with the use of serum or animal-derived substances for animal cell technology is the potential introduction of contaminants (adventitious agents) into the process and thus potentially into the final product . This fact led to an increased effort to replace serum-containing with serum-free media . In most cases, these media are supplemented with purified proteins, peptones, or hydrolysates, mainly of animal or human origin . Although such serum-free media are more defined than serum-containing media, the risk of the introduction of viruses by using animal-derived substances is still present, signifying that only a complete replacement of animal-derived substances by non-animal-derived products leads to a relatively safe serum-free medium . The potential replacement of these animal/human-derived substances by those of non-animal origin (e.g . plant origin) will be discussed . In several examples, the potential of serum-free media free of any animal-derived component in supporting cell growth and production of biologicals will be presented . In this context, the risk of using non-animal-derived substances in serum-free media for animal cell technology will be discussed with respect to classically used cell culture media.

Biochem Biophys Res Commun, 1999 Jul 14, 260(3), 734 - 9
Release of soluble ICAM-1 from human lung fibroblasts, aortic smooth muscle cells, dermal microvascular endothelial cells, bronchial epithelial cells, and keratinocytes; Leung KH; We determined effects of IL-1alpha, TNFalpha and IFNgamma on sICAM-1 release in culture media from human aortic smooth muscle cells (AOSMC), dermal microvascular endothelial cells (DMEC), keratinocytes (KC), bronchial epithelial cells (BEC) and lung fibroblasts (LF) as determined by ELISA . Under basal conditions of cultures for 20 h, low concentrations of sICAM-1 were only detected in the culture media of two (DMEC and BEC) of these cell types . IL-1alpha, TNFalpha and IFNgamma stimulated sICAM-1 from these cells . IFNgamma stimulated more shedding from AOSMC, BEC and KC than IL-1alpha or TNFalpha . TNFalpha enhanced more sICAM-1 release from DEMC than from AOSMC, BEC and LF . IL-1alpha and IFNgamma or TNFalpha and IFNgamma acted synergistically to enhance shedding of sICAM-1 from these cells . The levels sICAM-1 in pathophysiological conditions may influence leukocyte-vascular cell interactions to block leukocyte transmigration to tissue injury sites as a negative feedback mechanism .

Biochem Biophys Res Commun, 1999 Jul 5, 260(2), 534 - 9
L-Asparaginase inhibits the rapamycin-targeted signaling pathway; Iiboshi Y et al.; L-Asparaginase is widely used in the treatment of acute lymphoblastic leukemia . L-Asparaginase preparation derived from E . coli converts asparagine (Asn) and glutamine (Gln) to aspartate (Asp) and glutamate (Glu), respectively, and causes rapid depletion of Asn and Gln . It thus suppresses growth of malignant cells that are more dependent on an exogenous source of Asn and Gln than are normal cells . It remains unclear, however, which signaling events in leukemic cells are affected by L-asparaginase . Recently, amino acid sufficiency has been demonstrated to selectively regulate p70 S6 kinase (p70(s6k)) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), both of which are targeted by the anti-proliferative drug rapamycin . Here we demonstrate that addition of L-asparaginase to human leukemic cells inhibits activity of p70(s6k) and phosphorylation of 4E-BP1, but not activities of other cell growth-related serine/threonine kinases . The rate and kinetics of p70(s6k) inhibition by L-asparaginase were comparable to those seen by deprivation of Asn and/or Gln from cell culture media, suggesting that the effect of L-asparaginase on p70(s6k) is explained by depletion of Asn and/or Gln . Moreover, L-Asparaginase as well as rapamycin selectively suppressed synthesis of ribosomal proteins at the level of mRNA translation . These data indicate that L-asparaginase and rapamycin target a common signaling pathway in leukemic cells .

Mol Immunol, 1999 Feb, 36(3), 177 - 85
Serum starvation induced secondary V lambda J lambda rearrangement in a human plasma B cell line; Haruta H et al.; HB4C5 is a human antibody producing plasma B cell line that expresses the recombination activating gene-1 (RAG-1) and RAG-2 constitutively, but undergoes few secondary immunoglobulin gene rearrangements when cultured in fetal bovine serum-containing medium . Here, we found that depletion of serum from the culture media induces secondary VlambdaJlambda rearrangement in this cell line . To investigate the induction mechanism of secondary VlambdaJlambda rearrangement, we assessed the expression levels of RAG-1 and RAG-2 products, Vlambda germline transcription level and the amount of Vlambda signal broken ends (SBE) in HB4C5 cells cultured in serum-supplemented or serum-free medium . Western-blot analysis showed that the expression level for the RAG-1 and RAG-2 proteins was not affected by the serum depletion . Vlambda germline transcript was found to be constitutively expressed in HB4C5 cell line and this transcription level was not affected by the lack of serum . On the other hand, the amount of Vlambda SBE was shown to be increased in HB4C5 cells cultured in serum-free medium, suggesting that this increased formation of Vlambda SBE at least partly contributed to the enhanced occurrence of secondary VlambdaJlambda rearrangement in HB4C5 cells cultured in the serum-free condition . These results indicate that expression of RAG proteins and Vlambda germline transcription is not enough to undergo secondary VlambdaJlambda rearrangement in this cell line.

Hum Reprod, 1999 Jul, 14(7), 1819 - 22
The incidence of spontaneous acrosome reaction in homogeneous populations of hyperactivated human spermatozoa; Green S et al.; The incidence of spontaneous acrosome reaction occurring in 1314 individually selected hyperactivated (HA) human spermatozoa was compared to that occurring in 8226 individually selected non-hyperactivated spermatozoa (non-HA) sampled over an incubation time course to allow for capacitation . Two-way analysis of variance showed a significant difference between HA and non-HA spermatozoa for the mean percent acrosome reacted (R), partially acrosome reacted (PR) and combined total (R+PR) (P < 0.001) . One-way analysis showed that among the HA spermatozoa there were marked differences among the proportion showing R+PR at the various time points (P = 0.005) . Using the same end point, there was no significant evidence of change with time for the non-HA spermatozoa . The overall data indicated that HA human spermatozoa have a greater propensity for spontaneous acrosomal loss than non-HA spermatozoa during incubation in synthetic culture media.

Prostaglandins Other Lipid Mediat, 1999 Jun, 57(4), 189 - 205
PGE2 induces its own secretion in vitro by bovine 270-day placenta but not by 200-day placenta; Weems YS et al.; Two separate experiments were conducted to determine whether prostaglandin (PG) E2 stimulates the secretion of progesterone by 270- or 200-day Brahman placentas in vitro . Secretion of progesterone, PGF2alpha, pregnancy specific protein B, or estradiol-17beta by 270-day Brahman placentas was not affected (p > or = 0.05) by PGE2, during the 4-h incubation period at the doses tested . Indomethacin or meclofenamic acid decreased (p < or = 0.05) 270-day Brahman placental secretion of PGE and PGF2alpha by 98 and 60%, respectively . However, PGE2 induced (p < or = 0.05) its own secretion, but not the secretion of PGF2alpha (p > or = 0.05), by 270-day Brahman placentas, even in the presence of indomethacin or meclofenamic acid at a dose of 100 ng/mL . Also, secretion of 8-Epi-PGE2 by Day 270 Brahman placentas was increased (p < or = 0.05) by PGE2 . Secretion of progesterone, estradiol-17beta, or pregnancy specific protein B by 200-day Brahman placentas was not affected by PGE2, 8-Epi-PGE2, PGF2alpha, estradiol-17beta, or trichosanthin during the 4- or 8-h incubation period (p > or = 0.05) . Secretion of estradiol-17beta at 8 h was lower (p < or = 0.05) in all treatment groups and did not differ (p > or = 0.05) among the 8-h incubation treatment groups . Secretion of PGE by 200-day Brahman placentas was reduced (p < 0.05) by indomethacin 72 and 82% and by meclofenamic acid 72 and 96%, respectively, at 4 and 8 h when compared to controls . Secretion of PGF2alpha was reduced (p < or = 0.05) 71 and 86% by indomethacin or 89 and 89% by meclofenamic acid at 4 and 8 h, respectively, and did not differ (p > or = 0.05) between 4 and 8 h of incubation . PGE2 did not (p > or = 0.05) induce secretion of PGE above what was added in any treatment group . PGE in culture media was increased (p < or = 0.05) by 8-Epi-PGE2, pregnancy specific protein B, and the 100 ng/mL PGF2alpha dose (p < or = 0.05), but not by PGE2, progesterone, estradiol-17beta, 8-Epi-PGF2alpha, or trichosanthin . Secretion of PGF2alpha by 200-day Brahman placentas was not affected (p > or = 0.05) by 8-Epi-PGE2, progesterone, or estradiol-17beta, but PGF2alpha secretion was increased (p < or = 0.05) by trichosanthin or PGE2, even in the presence of indomethacin or meclofenamic acid . It is concluded that PGE does not affect secretion of progesterone by 200- or 270-day bovine placentas, but, pregnancy specific protein B may regulate placental secretion of PGE . Also, indomethacin and meclofenamic may affect enzymes converting PGH to PGE rather than acting only on cyclooxygenase because indomethacin and meclofenamic acid lowered PGE secretion by 270-day Brahman placentas more than they lowered PGF2alpha . In addition, it is concluded that PGE2 can induce bovine placental secretion of PGE, but this is dependent upon the stage of gestation.

Free Radic Biol Med, 1999 Jun, 26(11-12), 1457 - 66
Repair of iron-induced DNA oxidation by the flavonoid myricetin in primary rat hepatocyte cultures; Abalea V et al.; Oxidative DNA damage and its repair in primary rat hepatocyte cultures was investigated following 4 h of incubation with the toxic iron chelate, ferric nitrilotriacetate (Fe-NTA), in the presence or absence of the potent protective flavonoid myricetin (25-50-100 microM) . Seven DNA base oxidation products were quantified in DNA extracts by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode . Concomitantly, DNA repair capacity of hepatocytes was estimated by the release of oxidized-base products into culture media, using the same GC-MS method . A genotoxic effect of Fe-NTA (100 microM) in hepatocytes was evidenced by a severe increase in DNA oxidation over basal levels, with accumulation in cellular DNA of five oxidation products derived from both purines and pyrimidines . This prooxidant effect of iron was also noted by an induction of lipid peroxidation, estimated by free malondialdehyde production . Addition of increasing concentrations of myricetin (25-50-100 microM) simultaneously with iron prevented both lipid peroxidation and accumulation of oxidation products in DNA . Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media, especially of purine-derived oxidation products . This removal of highly mutagenic oxidation products from DNA of hepatocytes might correspond to an activation of DNA excision-repair enzymes by myricetin . This was verified by RNA blot analysis of DNA polymerase beta gene expression which was induced by myricetin in a dose-dependent manner . This represented a novel and original mechanism of cytoprotection by myricetin against iron-induced genotoxicity via stimulation of DNA repair processes . Since iron-induced DNA damage and inefficient repair in hepatocytes could be related to genotoxicity and most probably to hepatocarcinogenesis, modulation of these processes in vitro by myricetin might be relevant in further prevention of liver cancer derived from iron overload pathologies.

J Biomed Mater Res, 1999 Feb, 44(2), 176 - 90
In vitro osteoblastic differentiation of human bone marrow cells in the presence of metal ions; Morais S et al.; For periods up to 21 days human bone marrow was cultured in control conditions that favor the proliferation and differentiation of osteoblastic cells . The effect of AISI 316L corrosion products and the corresponding major separate metal ions (Fe, Cr, and Ni) were studied in three different phases of the culture period in order to investigate the effects of metal ions in cell populations representative of osteoblastic cells in different stages of differentiation . Toxicity consequences of the presence of metal ions in bone marrow cultures were evaluated by biochemical parameters (enzymatic reduction of MTT, alkaline phosphatase activity, and total protein content), histochemical assays (identification of ALP-positive cells and Ca and phosphates deposits), and observation of the cultures by light and scanning electron microscopy . Culture media were analyzed for total and ionized Ca and P and also for metal ions (Fe, Cr, and Ni) . The presence of AISI 316L corrosion products and Ni salt in bone marrow cultures during the first and second weeks of culture significantly disturbs the normal behavior of these cultures, interfering in the lag phase and exponential phase of cell growth and ALP expression . However, the presence of these species during the third week of culture, when expression of osteoblastic functions occurs (mineralization process), did not result in any detectable effect . Fe salt also disturbs the behavior of bone marrow cell cultures when present during the lag phase and proliferation phase, and a somewhat compromised response between the normal pattern (control cultures) and intense inhibition (AISI 316L corrosion products and Ni salt-added cultures) was observed . Fe did not affect the progression of the mineralization phase . Osteogenic cultures exposed to Cr salt (Cr3+) presented a pattern similar to the controls, indicating that this element does not interfere, in the concentration studied, in the osteoblastic differentiation of bone marrow cells . Quantification of metal ions in the culture media showed that Cr (originated from AISI 316L corrosion products but from not Cr3+ salt) and Ni (originated from AISI 316L corrosion products and Ni salt) appear to be retained by the bone marrow cultures .

Genes Chromosomes Cancer, 1999 Aug, 25(4), 316 - 22
HMGIC expression in human adult and fetal tissues and in uterine leiomyomata; Gattas GJ et al.; The high-mobility-group (HMG) protein gene, HMGIC, is localized to chromosome 12, band q15, a region often rearranged in benign mesenchymal tumors, including uterine leiomyomata . Although some evidence suggests a role in regulation of cell proliferation, the precise function of HMGIC in the development or progression of these tumors remains unclear . We investigated HMGIC expression in 17 fetal tissues (adrenal, aorta, bone, brain, heart, intestine, kidney, liver, lung, muscle, ovary, placenta, skin, spleen, stomach, testis, and uterus) and 10 adult tissues (aorta, brain, cerebellum, fat, kidney, liver, lung, lymph node, myometrium, and spinal cord) by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) assays . Comparisons between HMGIC gene expression in tumor samples from 11 uterine leiomyomata and 7 normal matched myometrium or in vitro cell cultures (chorionic villi, placenta, myometrium, leiomyoma, and skin) were also performed . The gene was expressed in all fetal tissues tested but only in adult lung and kidney . HMGIC was also expressed in leiomyoma tumor samples containing t(12;14) and in all in vitro cell cultures . The pattern of HMGIC expression suggests that this gene is important in rapidly proliferating human fetal tissues . Restoration of expression in leiomyomata required dysregulation of HMGIC . Transcripts of HMGIC can also be detected after in vitro cell culture, suggesting that HMGIC expression may be affected by factors present in culture media and serum . Genes Chromosomes Cancer 25:316-322, 1999 .

J Pathol, 1999 Mar, 187(4), 455 - 61
Renal cell carcinomas and pancreatic adenocarcinomas produce nidogen in vitro and in vivo; Oivula J et al.; The production of nidogen by four renal cell carcinoma (RCC) and three pancreatic adenocarcinoma (PAc) cell lines has been studied in cell culture and in xenografted tumours in nude mice . In RCC cells, immunoreactivity for nidogen was seen only after exposure to monensin to induce cytoplasmic accumulation of secretory proteins . In PAc cells, immunoreaction was also detectable in control cells . Immunoblotting of control and monensin-exposed cells and immunoprecipitation of culture media of radioactively labelled cells demonstrated the production of nidogen polypeptide of Mr ca . 150000 by six of the seven cell lines . Basement membranes (BMs) and stroma of the xenografted tumours derived from these six cell lines demonstrated immunoreactivity for both human and mouse nidogen, as revealed with species-specific antibodies . The ability of the cells to produce nidogen in vitro and deposit in vivo was positively correlated with high histological grade of the xenografted tumours, although the small number of cell lines studied calls for further studies to confirm this . The distribution of nidogen in human RCC and PAc specimens was also studied by immunohistochemistry . There was strong immunoreactivity for nidogen in tumour stroma, BM of carcinoma cell nests, and endothelial basal lamina, but no conclusions could be drawn regarding histological grade and immunostaining patterns, because stromal production could not be ruled out . The results show that nidogen is produced by human carcinoma cells both in vitro and in vivo .

Cancer Lett, 1998 Oct 23, 132(1-2), 175 - 80
The kinetics and cytotoxicity of cisplatin and its monohydrated complex; Yachnin JR et al.; This paper examines the monohydrated complex of cisplatin (MHC) with respect to kinetics and cytotoxicity . Equilibrium mixtures of cisplatin and hydrated species have been used in previous studies of a similar nature . To our knowledge, this is the first paper examining MHC after isolation and quantification . This was accomplished using liquid chromatography with porous graphitic carbon . MHC and cisplatin were quantified over time in a suspension of the small-cell lung cancer cell line U-1285 . Cytotoxicity was evaluated using the fluorescent microculture cytotoxicity assay . MHC was significantly more cytotoxic than cisplatin at the high end of the drug concentrations tested . In culture media with low chloride ion concentrations, the stability of MHC was related to changes in pH . At a pH of between 6.0 and 7.2, MHC was rapidly converted to cisplatin . In culture media with a pH above 7.2, MHC was considerably more stable . These findings might have clinical significance given that MHC circulates in the blood stream of patients receiving cisplatin infusions and that solid tumours often have environments that are extremely acidotic.

Med Biol Eng Comput, 1999 Mar, 37(2), 264 - 71
Dielectric single particle spectroscopy for measurement of dispersion; Schnelle T et al.; Measuring the frequency-dependent behaviour of single particles or biological cells in inhomogeneous and/or rotating electric fields is a sensitive method for characterising their dielectric properties . This technique is able to detect broad dispersion in the megahertz range of homogeneous artificial Sephadex G15 spheres . Recent progress has opened up the possibility of carrying out dielectric spectroscopy in cell culture media . Dielectrophoretic and electrorotational spectra of different cells in media of varying conductivity can only be explained by the introduction of dispersive cell compartments . The cytoplasm of animal cells typically exhibits a broad dispersion around 15 MHz and there is evidence for membrane dispersion around 50 MHz.

Neuroimmunomodulation, 1999 Jul-Aug, 6(4), 293 - 9
Effect of secretion of splenocytes after superior ovarian nerve section on the ovarian steroidogenesis; Forneris M et al.; It is known that ovary and spleen are innervated extensively by afferent and efferent noradrenergic sympathetic nerve fibers from the celiac ganglion . Furthermore, immune cells located in the ovary influence the ovarian physiology . However, the peripheral interaction between the immune and neuroendocrine system is poorly understood . This work was undertaken to study the effect of superior ovarian nerve (SON) transection, in adult rats, on the number of splenocyte beta-adrenergic receptors and their possible relation to ovarian steroidogenesis, measuring the effect of secretions of those splenocytes on progesterone and estradiol release from the ovary . Seven days after SON transection, the splenocytes were isolated and then cultured for 48 h . Their number of beta-adrenergic receptors, measured using {125I}-cyanopindolol as ligand, increased, and their culture media, used to stimulate ovaries from 60-day-old intact (neither SON-transected nor sham-operated) rats in vitro on diestrous day 2 showed a decrease in progesterone release and an increase in estradiol release in relation to splenocyte culture media of control rats (sham-operated; p < 0.001, respectively) . The effects of in vivo SON transection were simulated by an in vitro system modulating the splenocyte beta-adrenergic receptor number . The splenocytes from SON-transectioned rats were preincubated with and without norepinephrine (NE) 10(-6) M for 48 h, a low and high number of beta-adrenergic receptors respectively, and then were stimulated with NE 10(-6) M for 24 h . After that, the culture medium from splenocytes with a low number of beta-adrenergic receptors induced progesterone release from the ovaries of intact rats (p < 0 . 001), but produced no change in estradiol release . The data suggest that splenocyte secretions, which participate in the ovarian steroidogenic response, particularly in progesterone release, might be controlled by adrenergic influences since the number of splenocyte beta-adrenergic receptors changes through SON-celiac ganglion-noradrenergic postganglionic innervation of the spleen . In estradiol release, probably other neurotransmitters than norepinephrine (NE) are involved when the SON is sectioned . In this paper we also show functional evidence for modulation of immune function by the sympathetic nervous system and its principal neurotransmitter, NE.

J Biochem (Tokyo), 1999 Jul, 126(1), 10 - 8
Hyperproduction of recombinant ferredoxins in escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster; Nakamura M et al.; Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism . To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli . Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step . We examined the coexpression of the E . coli ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein {Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R . (1998) J . Biol . Chem . 273, 13264-13272}, with reporter Fds using compatible plasmids . The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed . The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 x YT medium or Terrific broth, and with or without supplemental cysteine or iron . These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.

Diabetes, 1999 Jul, 48(7), 1409 - 14
Enhancing effects of long-term elevated glucose and palmitate on stored and secreted proinsulin-to-insulin ratios in human pancreatic islets; Bjorklund A et al.; Relative hypersecretion of proinsulin is a feature of type 2 diabetes . We investigated to what extent this feature can be induced in human pancreatic islets by elevated glucose or fatty acids, two major abnormalities of the diabetic state . A 48-h culture period with 27 mmol/l glucose increased the intraislet proinsulin-to-insulin (PI/I) ratio 5.0-fold, owing to preferential decrease of insulin . The PI/I ratio in culture medium was enhanced 1.9-fold versus islets cultured with 5.5 mmol/l glucose . This effect of elevated glucose persisted after normalization of glucose levels: during 60-min postculture incubations at a basal glucose concentration (3.3 mmol/l), the PI/I ratio of secretion increased 4.9-fold . The ratio was also increased (14-fold) after renewed postculture stimulation with 16.7 mmol/l glucose . Diazoxide was added to culture medium to block glucose-induced insulin secretion and thus investigate the importance of overstimulation . In cultures at 27 mmol/l glucose, the presence of diazoxide decreased the PI/I ratio of islet contents by 76%, the accumulated secretion to culture medium by 70%, and the release at 3.3 or 16.7 mmol/l glucose during postculture incubations by 85 and 86%, respectively . None of these PI/I-decreasing effects of diazoxide were reproduced during or after coculture with 5.5 mmol/l glucose . Culture with 0.2 mmol/l palmitate and 5.5 mmol/l glucose decreased islet contents of proinsulin and insulin and increased the secreted products in culture media without affecting PI/I ratios . During postculture conditions, however, prior palmitate culture enhanced the PI/I ratio of release at 3.3 mmol/l glucose (from 2.2 +/- 0.4 to 5.4 +/- 0.9%, P < 0.05) . Culture with palmitate together with 27 mmol/l glucose decreased islet contents of proinsulin and insulin and further enhanced intraislet PI/I ratios (from 9.3 +/- 1.1 to 13.4 +/- 2.5%, P < 0.05) . However, palmitate failed to affect PI/I ratios in culture medium . In contrast, in postculture incubations at 3.3 mmol/l glucose, prior palmitate culture further elevated the PI/I ratio of secretion (from 10.8 +/- 1.2 after previous 27 mmol/l glucose alone to 13.9 +/- 2.8% after palmitate and glucose, P < 0.05) . We conclude that 1) long-term exposure of human islets to elevated glucose leads to preferential secretion of proinsulin, and this effect persists also after glucose normalization; 2) the glucose effect appears secondary to depletion of mature insulin granules; and 3) elevated fatty acids influence PI/I ratios of secretion by mechanisms that are, in part, incongruous with an over-stimulation effect.

Toxicol Appl Pharmacol, 1999 Jul 1, 158(1), 61 - 70
Quantitative measurement of acute corneal injury in rabbits with surfactants of different type and irritancy; Maurer JK et al.; We have hypothesized that differences in ocular irritancy are related to differences in extent of initial injury and that, regardless of the processes leading to tissue damage, extent of injury is the primary factor that determines the final outcome of ocular irritation . In previous in vivo confocal microscopic (CM) studies we identified quantifiable differences in the extent of corneal injury occurring with four surfactants (three anionic, one cationic) known to cause different levels of ocular irritation and demonstrated that extent of initial corneal injury was related to the magnitude of cell death . The purpose of this study was to assess the applicability of this hypothesis to a broad sampling of surfactants . Specifically, initial corneal changes induced by seven different surfactants (one anionic, three cationic, three nonionic) were measured by in vivo CM and cell death was measured by an ex vivo live/dead assay . The right eye of each rabbit was treated by placing 10 microl of a surfactant directly on the cornea . Eyes were examined macroscopically and scored for irritation at 3 h and 1 day . At 3 h and 1 day, in vivo CM was used to examine the corneas and quantitate epithelial cell size, epithelial thickness, corneal thickness, and depth of stromal injury . At 3 h and/or at 1 day, corneas were removed and excised regions were placed in culture media containing 2 microM calcein AM and 4 microM ethidium homodimer . Using laser scanning CM, the number of dead epithelial and/or stromal cells in a 300 x 300 x 170-microm3 (xyz) volume of the cornea was determined . In vivo CM and live/dead assay findings revealed three surfactants to affect only the epithelium, three surfactants to affect the epithelium and superficial stroma, and one surfactant to affect the epithelium and deep stroma . Extent of initial corneal injury reflected level of ocular irritation, and magnitude of cell death was related to the extent of initial corneal injury . These findings are consistent with those for known slight, mild, and moderate to severe irritants, respectively . They suggest that our hypothesis is broadly applicable to surfactants . Additionally, we believe these surfactants should be included as part of a new "gold standard" for use in developing and validating in vitro tests to replace the use of animals in ocular irritancy testing .

FEMS Microbiol Lett, 1999 Jun 15, 175(2), 217 - 21
Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts; Turrens JF et al.; The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease . This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T . cruzi (ID50 = 35 microM) . When added to intact cells, MPNO inhibited the growth of T . cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T . cruzi trypomastigotes (ID50 = 20 microM) . At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts . Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO . Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography . Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.

Atherosclerosis, 1999 May, 144(1), 123 - 34
A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning; Voisard R et al.; BACKGROUND: Restenosis is a reparative process that is activated in response to injury induced by angioplasty . Despite numerous experimental models of restenosis the number of human arterial organ culture systems is very limited and long-term experiences do not exist . METHODS AND RESULTS: During routine nephrectomies parts of the renal arteries of 88 patients were extracted, 47 were suitable for organ culture preparations . Sections were made at 3 mm intervals perpendicular to the vessel wall axis . The arterial segments were treated with 3 mm standard balloon-catheters (Medtronic 14K2030E) for 60 s with 3, 6, 9, and 12 bar . After angioplasty, the organ segments were cultured in a mixture of Waymouth's MB 752/1 and Ham F-12, supplemented with 15% fetal calf serum . After 0, 4, 14, 21, 28, and 56 days the organ cultures were fixed in 4% para-formaldehyde and embedded in paraffin . After staining with a modified elastica-van Gieson technique the intimal wall thickening was analyzed with a computerized morphometric system . For the identification of smooth muscle cells (SMC) a monoclonal antibody against smooth muscle alpha-actin was used . Endothelial cells were identified using an anti-human von Willebrand factor . To determine the number of cells undergoing DNA synthesis, bromodeoxyuridine (BrdU), a thymidine analogue, was added to the culture media 18 h prior to fixation . BrdU was detected with a monoclonal antibody, as secondary antibody a biotinylated horse-anti-mouse antibody was used . After 14, 21, and 28 days in culture BrdU-positive cells were detected in the neointima of the organ cultures, indicating mitotic activity in this area . After 28 and 56 days in culture a clear increase of neointimal thickening was found in the morphometric analysis . By positive reaction with antibodies against smooth muscle alpha-actin these cells were partly identified as SMC . CONCLUSIONS: The organ culture model offers opportunities for in vitro investigations of postangioplasty restenosis . The data emphasize the importance of a relatively late proliferative response of SMC in the human arterial organ culture model.

Exp Clin Endocrinol Diabetes, 1999, 107(3), 214 - 9
Human pancreatic islet quality control: easy assessment of metabolic functions; Vandewalle B et al.; We describe simplified and rapid methods to assess islet function with the aim to develop better protocols for islet isolation and to determine islet characteristics before transplantation . These methods are also useful in the assessment of the potentially beneficial or deleterious effects of compounds added to the culture media in stimulation experiments . To this end, we took advantage of the multiscreen assay system produced by Millipore SA . This 96-well unit allowed the free-floating culture of islets on filter membranes, the rapid vacuuming and collection of conditioned media or reaction buffer and thus successive testing of the same number of islets, possibly at different culture times . We estimated islet viability by determination of the metabolic activity of cells, normal function of islets by their ability to metabolize glucose and to synthesize and secrete insulin and of nitrite release, a reflection of nitric oxide (NO) status of cells, which may be involved in a signaling pathway during glucose-stimulated insulin secretion or in cytokine inducible pathway . Assays may be performed either on selected islets or on aliquots of semi-purified preparations designated for grafting, allowing thus the rapid estimation of graft function of the entire preparation . This herein described system may be also extended to many other functional tests.

Brain Res, 1999 Jul 3, 833(2), 202 - 8
Acetaldehyde cytotoxicity in cultured rat astrocytes; Holownia A et al.; The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both 'in vivo' and 'in vitro' ethanol exposure . Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells . Acetaldehyde levels in the culture media attained concentrations of approximately 450 microM . To study ethanol effects, alcohol oxidation was inhibited by 4-methylpyrazole (an inhibitor of alcohol dehydrogenase) . Acetaldehyde but not ethanol increased intracellular calcium levels by 155% . Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis . Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected . Nitrate levels in the culture media were unchanged . Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde . It can be concluded, that in 'in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated .

Zhonghua Yu Fang Yi Xue Za Zhi, 1998 Nov, 32(6), 336 - 9
{Studies on effects of cytokine released from alveolar macrophage induced by mineral dust on lung fibroblast}; Zhou J et al.; OBJECTIVE: To understand the role of cytokine released from alveolar macrophage (AM) in lung fibrosis caused by mineral dust . METHODS: Rabbit's AM obtained by lavage was cultured with mineral dust in vitro . Activities of tumor necrosis factor (TNF) and interleukin 6 (IL-6) in its supernatant were determined with isotope labelling method and MTT colorimetry, respectively . Human fetal lung fibroblast WI-138 was cultured with this supernatant . Proliferation of fibroblast and synthesis of collagen were examined by 3H-thymidine (3H-TdR) and 14C-proline (14C-Pro) incorporation and its total hydroxyproline (HOP) level was analyzed by chloramine-T method . RESULTS: Proliferation of lung fibroblast and synthesis of collagen could be enhanced by the supernatant containing AM induced by quartz, asbestos and uranium mineral dust, with 3H-TdR incorporated counts per minute (cpm) of 6,584, 3,848 and 6,893 in the group of 100 micrograms 3H-TdR and 14C-Pro incorporated 27,952, 13,416 and 18,538 in the group of 200 micrograms respectively, which were significantly higher than those in the control group . Total HOP levels in the culture media for lung fibroblast enhanced by AM supernatant were 22.41, 24.00 and 21.39 micrograms/ml, respectively, and was significantly higher than that in the control group (12.91 micrograms/ml) . Release of TNF and IL-6 could be stimulated by mineral dust, such as quartz, asbestos and uranium mineral dust, and their activities were significantly higher than those in the control group, with those of 1,396, 1,198 and 852 U/ml in TNF group and 1,336, 1511 and 1,335 U/ml in IL-6 group, respectively . Proliferation of lung fibroblast and synthesis of collagen could be inhibited by antibody against TNF and interferon-r (gamma), and the effect of the latter was weaker than that of the former on inhibition of fibroblast proliferation and the effect on collagen synthesis was just in the opposite direction . CONCLUSION: Lung fibrosis caused by mineral dust correlated with abnormal expression of TNF and IL-6 . Antibody against TNF and gamma interferon could antagonize the effect of NTF and IL-6.

Zhonghua Bing Li Xue Za Zhi, 1997 Oct, 26(5), 281 - 4
{The inhibitory effect of combined treatment of TNF alpha and antisense oligodeoxynucleotides on the growth of human pancreatic carcinoma cell line cells}; Zhang L et al.; OBJECTIVE: To study the inhibitory effect induced by combined treatment of TNF alpha and antisense oligodeoxynucleotide on the growth of human pancreatic adenocarcinoma cell line (PC-2) cells . METHOD: TNF alpha and synthesized antisense c-myc or Ki-ras phosphorothioate oligodeoxynucleotide (ASPODN) were added to the culture media of PC-2 cells, whereas the groups treated with TNF alpha or ASPODN alone were used as controls . The cell growth rate was estimated by cell count and MTT analysis, the endogenous target gene expression was studied by adopting RT-PCR-Southern blot technique, and cell apoptosis was detected in situ . RESULTS: The cell growth was inhibited much more obviously in the groups of combined treatment than in the groups treated with TNF alpha or ASPODN alone . Marked inhibition of endogenous Ki-ras and c-myc expression was observed in the groups treated with Ki-ras and c-myc ASPODN . The amounts of apoptotic cells were 8.0% and 8.4% in the groups of TNF alpha + Ki-ras ASPODN and TNF alpha + c-myc ASPODN, and were 5.2%, 4.8% and 5.4% for TNF alpha, Ki-ras ASPODN and c-myc ASPODN respectively . CONCLUSION: Our results demonstrate that the inhibitory effect induced by combined treatment of TNF alpha and ASPODN on cell growth was stronger than that induced by TNF alpha or ASPODN alone.

J Physiol, 1999 Jul 1, 518 ( Pt 1), 257 - 69
Interstitial cells of cajal generate electrical slow waves in the murine stomach; Ordog T et al.; 1 . The gastric corpus and antrum contain interstitial cells of Cajal (ICC) within the tunica muscularis . We tested the hypothesis that ICC are involved in the generation and regeneration of electrical slow waves . 2 . Normal, postnatal development of slow wave activity was characterized in tissues freshly removed from animals between birth and day 50 (D50) . Slow wave amplitude and frequency increased during this period . Networks of myenteric ICC (IC-MY) were present in gastric muscles at birth and did not change significantly in appearance during the period of study as imaged by confocal immunofluorescence microscopy . 3 . IC-MY networks were maintained and electrical rhythmicity developed in organ culture in a manner similar to normal postnatal development . Electrical activity was maintained for at least 48 days in culture . 4 . Addition of a neutralizing antibody (ACK2) for the receptor tyrosine kinase, Kit, to the culture media caused progressive loss of Kit-immunoreactive cells . Loss of Kit-immunoreactive cells was associated with loss of slow wave activity . Most muscles became electrically quiescent after 3-4 weeks of exposure to ACK2 . 5 . In some muscles small clusters of Kit-immunoreactive IC-MY remained after culturing with ACK2 . These muscles displayed slow wave activity but only in the immediate regions in which Kit-positive IC-MY remained . These data suggest that regions without Kit-immunoreactive cells cannot generate or regenerate slow waves . 6 . After loss of Kit-immunoreactive cells, the muscles could not be paced by direct electrical stimulation . Stimulation with acetylcholine also failed to elicit slow waves . The data suggest that the generation of slow waves is an exclusive property of IC-MY; smooth muscle cells may not express the ionic apparatus necessary for generation of these events . 7 . We conclude that IC-MY are an essential element in the spontaneous rhythmic electrical and contractile activity of gastric muscles . This class of ICC appears to generate slow wave activity and may provide a means for regeneration of slow waves.

Prostaglandins Other Lipid Mediat, 1999 Jan, 57(1), 1 - 12
Effects of angiogenic growth factors on endothelium-derived prostacyclin production by ovine uterine and placental arteries; Krishnamurthy P et al.; Uteroplacental and fetoplacental arteries produce substantial amounts of prostacyclin (PGI2) . Because angiogenic growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) are increased in pregnancy, we hypothesized that treatment of uterine and fetoplacental arteries with bFGF, VEGF, and EGF would further enhance the pregnancy-induced increase in PGI2 production . Duplicate uterine (UA) and fetoplacental (PA) artery (primary branch off of the umbilical cord = pPA; cotyledonary or tertiary = tPA) explants from seven late gestation sheep were placed in tissue culture (RPMI; 37 degrees C) for 24 h alone or with (1-100 ng/mL) bFGF, VEGF, or EGF . To evaluate the endothelial contribution to basal and stimulated PGI2 production and to determine whether it is de novo, arteries with and without endothelium from three additional late gestation ewes, tissues were incubated in the absence or presence of growth factors with or without meclofenamate (1 microM) . The stable metabolite of PGI2, 6-keto-PGF1 alpha, was measured in culture media and expressed as ng/mg wet wt 24 h . PGI2 production by UA increased (p < 0.05) from 5.43 +/- 0.26 at control to 8.93 +/- 0.99 with 100 ng/mL bFGF . Although VEGF produced a similar response, EGF did not increase PGI2 production in UA . In pPA, 100 ng/mL bFGF induced a 2.2-fold increase (p < 0.01) in PGI2 production from 1.94 +/- 0.14 to 4.20 +/- 0.31; VEGF and EGF were without effect . In tPA, 50 and 100 ng/mL bFGF increased PGI2 production from 1.98 +/- 0.14 to 3.5 +/- 1.05 and 3.96 +/- 0.46 (p < 0.02) . In tPA, VEGF did not increase PGI2 production; however, 10, 50, and 100 ng/mL EGF, enhanced (p < 0.03) PGI2 production from 1.98 +/- 0.14 to 3.39 +/- 0.62, 3.62 +/- 0.26, and 2.93 +/- 0.20 . Endothelium removal and meclofenamate treatment caused a 90% and 100% decrease, respectively, in basal PGI2 production, with no recovery after treatment with growth factors . We conclude that PGI2 production is augmented by bFGF in UA, pPA and tPA, by VEGF in UA, and by EGF in tPA during ovine pregnancy . Basal and stimulated PGI2 secretion is endothelium-derived via de novo synthesis . bFGF, VEGF, and EGF, in addition to angiogenesis, may modulate PGI2 production, further enhancing blood flow to the growing uteroplacental bed.

J Hepatol, 1999 May, 30(5), 868 - 75
The Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat hepatic stellate cells; Svegliati-Baroni G et al.; BACKGROUND/AIMS: Oxidative stress is associated with liver fibrosis in vivo and with hepatic stellate cell (HSC) activation in vitro, but the intracellular mechanisms mediating these effects are mostly unknown . The Na+/H+ exchanger plays a key role in regulating the cell cycle, and is involved in HSC proliferation . Its role in different HSC features, such as collagen accumulation, is still unknown . We thus evaluated if the Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat HSC . METHODS: HSC were incubated with 0.1 mM ferric nitrilotriacetate complex (FeNTA) . Intracellular hydroperoxides and malonildialdehyde (MDA) levels in the culture media were measured by the dichlorofluorescein and TBARS method, respectively . Intracellular pH and Na+/H+ exchanger activity were measured using the fluorescent dye BCECF . Cell proliferation was measured by immunohistochemistry for bromodeoxyuridine incorporation . Collagen type I accumulation in the culture media was measured by ELISA . RESULTS: HSC incubation with FeNTA resulted in a significant production of intracellular hydroperoxides and MDA, associated with increased Na+/H+ exchange activity and baseline intracellular pH (pHi) . Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium . All these effects were reversed by the antioxidant resveratrol and by the Na+/H+ exchanger inhibitor amiloride . CONCLUSIONS: This study indicates that the Na+/H+ exchanger might represent a common mediator of the different effects induced by oxidative stress on HSC . The reduction in cell proliferation and collagen synthesis induced by amiloride could represent a new therapeutic challenge in liver fibrosis.

J Clin Microbiol, 1999 Jul, 37(7), 2369 - 70
Delayed versus immediate bedside inoculation of culture media for diagnosis of vaginal trichomonosis; Schwebke JR et al.; A comparison of delayed versus immediate inoculation of culture medium for the diagnosis of trichomonosis was conducted . The sensitivities of the two methods were 100 and 97.4%, respectively . Delayed inoculation of culture medium for women without evidence of trichomonosis on direct microscopic examination is a valid diagnostic procedure.

J Clin Microbiol, 1999 Jul, 37(7), 2350 - 1
Inactivation of Mycobacterium tuberculosis for DNA typing analysis; Bemer-Melchior P et al.; DNA fingerprinting analysis of Mycobacterium tuberculosis is used for epidemiological studies and the control of laboratory cross-contamination . Because standardized procedures are not entirely safe for mycobacteriology laboratory staff, the paper proposes a new technique for the processing of specimens . The technique ensures the inactivation of M . tuberculosis before DNA extraction without the loss of DNA integrity . The control of inactivated cultures should be rigorous and should involve the use of two different culture media incubated for at least 4 months.

FEMS Microbiol Lett, 1999 Jun 1, 175(1), 79 - 85
The Mycobacterium marinum G13 promoter is a strong sigma 70-like promoter that is expressed in Escherichia coli and mycobacteria species; Barker LP et al.; A Mycobacterium marinum promoter, designated G13, was isolated from a promoter-trap library as a constitutive producer of the mutant green fluorescent protein . Sequence analysis, primer extension analysis, and computer promoter prediction analysis indicate that the G13 promoter is very similar to Escherichia coli consensus sigma 70 promoters . Expression of the green fluorescent protein from the G13 promoter in M . marinum is, however, up to 40 times higher than that seen from the mycobacterial hsp60 promoter during exponential growth . Further, expression from this promoter does not appear to affect the growth of the organism in culture media or in macrophages . The strong expression of the G13 promoter allows it to be developed as a useful molecular tool for high level expression of markers in vitro.

Invest New Drugs, 1998-99, 16(3), 199 - 204
Cryptophycin 1 cellular levels and effects in vitro using L1210 cells; Foster BJ et al.; Cryptophycin 1 is a natural product that was initially isolated from blue-green algae which has shown potent broad spectrum antitumor activity in preclinical in vitro and in vivo models . The drug strongly binds to tubulin and disrupts microtubule assembly for more than 24 hours after its removal . We evaluated cell survival, intracellular levels and inhibition of macromolecular synthesis in L1210 cells following exposure to cryptophycin 1 in vitro . Cell survival was strongly inhibited following drug exposure for either 1 or 4 hours . Intracellular drug levels were minimally affected by temperature (4 degrees C versus 37 degrees C) or exposure times up to 1 hour . However, extracellular drug concentration in culture media and increasing cell numbers did affect the concentration of intracellular drug levels in a nearly proportional manner . The synthesis of DNA and RNA was inhibited less than 5%, while protein synthesis inhibition was near 30% . Thus, none of the macromolecules were inhibited enough to explain the inhibition of tumor cell growth.

J Neurooncol, 1999 Mar, 42(1), 35 - 44
Growth inhibition and radiosensitization of cultured glioma cells by nitric oxide generating agents; Kurimoto M et al.; The authors examined the effect of nitric oxide (NO) generating agents on the growth and radiosensitivity of cultured glioma cells . Three glioma, rat C6, and human T98G and U87 cell lines were treated with the NO generating agents, S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP) . These agents released NO in the cell culture media and inhibited the growth of the glioma cells . Growth-inhibition was attenuated by hemoglobin, a known inhibitor of NO, suggesting it is mediated by NO . When C6 and T98G cells were irradiated in the presence of SNAP or SNP at 100 microM, radiosensitization was observed . SNAP at 100 microM exhibited a sensitizer enhancement ratio (SER) of 1.4 for C6 cells and 1.8 for T98G cells . SNP at 100 microM only radiosensitized T98G cells with a SER of 1.9 . The effect of SNP on radiosensitization of C6 cells was unclear . We conclude that NO generating agents are potential growth inhibitors and radiosensitizers for malignant glioma cells . NO mediated radiosensitization of glioma cells by NO generating agents may offer a new therapeutic approach for malignant glioma.

J Pediatr Surg, 1999 May, 34(5), 774 - 9; discussion 780
Epithelio-mesenchymal interactions in the developing mouse pancreas: morphogenesis of the adult architecture; Rose MI et al.; BACKGROUND/PURPOSE: The mammalian pancreas is thought to develop through a complex interaction between the budding epithelium and the surrounding mesenchyme . The exact nature of this interaction is unclear . Most of what is known to date of these interactions comes from a series of organ culture experiments done in the late 1960s . Nevertheless, these important experiments may have been confounded by less-defined culture media and organ dissection techniques, because the results are not reproducible in our hands . The authors undertook a study to reexplore these basic epithelio-mesenchymal interactions . METHODS: Using previously described organ dissection and culture techniques the authors examined the basic interactions between the embryonic pancreatic epithelium and its mesenchyme with histological and immunohistological techniques . RESULTS: The authors found that, contrary to previous reports, the earliest pancreatic anlage did not possess the intrinsic signaling necessary to support normal growth and differentiation in vitro . Intimate contact between the epithelium and the mesenchyme may be necessary until E11.5 for normal growth and differentiation . The age of the mesenchyme seemed to correlate with the degree of acinar differentiation, and proximity of mesenchyme was important for acinar differentiation . CONCLUSIONS: Previous investigations into the basic epithelio-mesenchymal interactions in the developing mammalian pancreas may have had confounding factors . Extrinsic signals seem necessary for complete pancreatic differentiation, and mesenchymal factors appear important for acinar differentiation.

Jpn J Cancer Res, 1999 Mar, 90(3), 308 - 19
High-molecular-weight fibronectin synthesized by adenoid cystic carcinoma cells of salivary gland origin; Toyoshima K et al.; To understand the morphogenesis of characteristic cribriform structures and the frequent invasion of salivary adenoid cystic carcinomas (ACC) along such basement membrane-rich structures as peripheral nerves, we have isolated fibronectin (FN) from the culture media of ACC3 cells established from a parotid ACC and characterized its glycosylation and alternative splicing status . FN isolated from ACC3 cells (ACC-FN) showed a molecular mass of 315 kDa in SDS-PAGE and was less heterogeneous and larger than plasma FN (pFN) or FNs from other cell sources . Differential enzymatic treatments of immunoprecipitated ACC-FN with neuraminidase, peptide-N-glycosidase F and endo-alpha-N-acetylgalactosaminidase revealed that ACC-FN was composed of a polypeptide chain of 270 kDa, with 10 kDa each of N-linked and O-linked oligosaccharide chains . Reverse transcription polymerase chain reaction (RT-PCR), in-situ hybridization, and immunofluorescence studies showed that most ACC-FNs contained ED-A, ED-B and IIICS regions in the molecules . This alternative splicing status of ACC-FN seemed to contribute to its less heterogeneous and larger molecular form . Cell attachment assay demonstrated that ACC-FN was more potent than pFN in adhesion of ACC3 cells . The results indicated that ACC-FN may function as a substrate for attachment of ACC3 cells, or that ACC3 cells trap and retain ACC-FN in their pericellular space . This isoform of FN may play an important role in the mode of invasion of ACC and the formation of stromal pseudocysts in the characteristic cribriform structure of ACC.

Hum Reprod, 1999 Jun, 14(6), 1555 - 62
Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles; Wright CS et al.; In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development . Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis . An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro . Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days . Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained . The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability . Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture . The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles . Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone . Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.

Biotechnol Prog, 1999 May-Jun, 15(3), 373 - 82
Model simulation and analysis of perfusion culture of mammalian cells at high cell density; Zeng AP et al.; Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures . Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability . Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody . Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate . This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered . It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate . In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies . Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed . For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high . The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration . The mathematical model can be used to predict these optimum conditions and may also be used for process design.

Biotechnol Prog, 1999 May-Jun, 15(3), 336 - 46
Stable production of a human growth hormone antagonist from CHO cells adapted to serum-free suspension culture; Haldankar R et al.; Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kDa . An hGH analogue was created with a single amino acid substitution (glycine{G} 120 to arginine{R}) in the third alpha-helix of the hGH molecule . This hGH analogue, named hGHG120R, was found to be an hGH antagonist . It is a parenteral drug candidate for treating conditions in which hGH levels are abnormally high, as found in type I diabetics . Previously, a genetically engineered anchorage-dependent mouse L cell line was created that produced and secreted hGHG120R in culture media (Dulbecco's modified Eagle's medium, DMEM) supplemented with 5% NuSerum IV . A multistep downstream process was developed to purify hGHG120R . The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, size exclusion chromatography, reversed phase high-performance liquid chromatography, phase separation, and lyophilization . Here, we present the development of a superior eukaryotic system using a proper combination of genetic elements, cell line, and media formulation . This system is suitable for the large-scale production of the recombinant protein and is superior to the previously developed system in that it increases the specific production rate and at the same time eases the burden of the purification process, in both time and efficiency . Dihydrofolate reductase mutant (DHFR-) Chinese hamster ovary (CHO) cells were used that were stably transfected with an expression vector in which the hGHG120R gene is driven by the relatively strong human cytomegalovirus-early gene regulatory region . The hGHG120R tested to be biologically active . These cells were then adapted to grow in suspension in CHO-S-SFM (serum-free media) . High cell densities, typically 2.0 x 10(6) cells/mL were obtained from spinner flask cultures . Partial purification of hGHG120R from CHO cell cultured media revealed that the level of impurities in SFM was significantly lower than the serum-supplemented DMEM . This suggests that the salt precipitation and the SEC step need not be employed in the purification of hGHG120R from SFM . This would result in a reduction of the operating time by 50 h and boost the recovery yield of hGHG120R to 75%.

J Bone Miner Res, 1999 Jun, 14(6), 904 - 14
Two human osteoblast-like osteosarcoma cell lines show distinct expression and differential regulation of parathyroid hormone-related protein; Jemtland R et al.; Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC) . However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood . Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM) . Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines . PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells . Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines . In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression . Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect . In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA . In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively . PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells . The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line-specific defects . Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.

Hepatology, 1999 Jun, 29(6), 1743 - 51
Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways; Svegliati-Baroni G et al.; Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells . IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC) . This study evaluates the potential roles of these hormones in the development of liver fibrosis . Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue . Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively . Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis . Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation . Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin . Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway . IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism . In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.

Osteoarthritis Cartilage, 1998 Nov, 6(6), 435 - 40
Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts; Dodge GR et al.; OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein . Each of its five subunits is approximately 100,000 Da in molecular weight . COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon . To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture . METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent . During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium . The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies . The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels . For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined . RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts . COMP could be easily detected by immunoprecipitation in all cell types . Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used . COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells . The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively) . CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts . These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.

Osteoarthritis Cartilage, 1998 Nov, 6(6), 427 - 34
Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro; Bassleer C et al.; OBJECTIVE: This study investigated the in-vitro effects of a crystalline glucosamine sulfate (GS) preparation on DNA synthesis and on proteoglycan (PG) and type II collagen (coll II) production by human articular chondrocytes isolated from human osteoarthritic articular cartilage in a 3-dimensional culture system for 4, 8, and 12 days . MATERIALS AND METHODS: Human articular chondrocytes from osteoarthritic femoral heads were isolated from their matrix by collagenase digestion and then cultured in suspension . Under constant agitation, cells aggregated and formed a cluster within a few days . The effects of GS (1-100 micrograms/ml) on chondrocytes were determined by quantifying DNA synthesis (by measurement of {3H}-thymidine uptake) as well as PG and coll II production using radiommunoassays (RIAs) specific for coll II and to human human cartilage PG . Cross-reaction with GS in the RIAs was not detected . Moreover, PG size distribution was determined by exclusion chromatography under associative conditions to determine the association of PG monomers with hyaluronic acid (HA) to form large molecular weight PG aggregates . RESULTS: Under the above conditions, PG production in culture media and chondrocyte clusters was increased by GS (10-100 micrograms/ml) . DNA synthesis and coll II production were not modified by GS . In addition, GS did not modify the physico-chemical form of PG produced by cells during culture . CONCLUSIONS: Glucosamine sulfate did not affect DNA synthesis nor coll II production but caused a statistically significant stimulation of PG production by chondrocytes from human osteoarthritic cartilage cultured for up to 12 days in 3-dimensional cultures.

Endocrinology, 1999 Jun, 140(6), 2549 - 54
Dimeric inhibin A and B production are differentially regulated by hormones and local factors in rat granulosa cells; Lanuza GM et al.; In the present study, we have examined the role of hormones and growth factors in regulating dimeric inhibin production in immature rat granulosa cells . Purified granulosa cells from estrogen-primed immature rats were cultured under defined conditions . Inhibins A and B in the culture media were measured using a two-site enzyme-linked immunosorbent assay specific for each dimer . Under basal conditions, granulosa cells produced 14-fold more inhibin A than inhibin B (inhibin A, 2.0; inhibin B, 0.14 ng/ml, measured against human standards; average A/B apparent ratio, 14) . Addition of increasing doses of FSH elicited dose-dependent increases in both inhibins, the effects being more pronounced on inhibin A than on inhibin B (9.4- and 4.1-fold increases, respectively; average A/B ratio, 34) . Estradiol, when added alone, stimulated inhibin A production 3- to 6-fold, whereas minor changes were observed in inhibin B production . Insulin-like growth factor-I produced a similar stimulation of both inhibins (3-fold stimulation over control) . This growth factor, however, induced a marked dissociation in the sensitivity of inhibins A and B to FSH stimulation, with maximal stimulation of inhibin B observed at comparatively lower concentrations of the gonadotropin . Transforming growth factor-beta (TGF-beta, 5 ng/ml) had a more marked stimulatory effect on inhibin B than on inhibin A production (7- to 14-fold vs . 2- to 5-fold for inhibin B and A, respectively) . A more pronounced differential stimulation of inhibin B was also exerted by another member of the TGF-beta superfamily, activin A (A/B ratio, 0.66) . This preferential stimulation of inhibin B by TGF-beta and activin A was amplified in the presence of FSH . Coculture of rat granulosa cells with freshly isolated bovine oocytes was also associated with a marked stimulation of inhibin B production (100-fold increase) and a comparatively lower stimulation of inhibin A (10-fold increase; A/B ratio, 1) . The discrepancy between the proportion of inhibin dimers in serum (A/B ratio, 0.13) and those produced by untreated granulosa cells may suggest that intraovarian factors, such as TGF-beta, activin A, or oocyte-derived factor(s), are responsible for the shift of the ratio toward the predominance of inhibin B.

Mol Hum Reprod, 1999 Jun, 5(6), 548 - 53
Platelet-activating factor stimulates cytokine production by human endometrial stromal cells; Nasu K et al.; Although preimplantation embryo and decidual cells secrete significant amounts of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF); its precise function in early pregnancy has yet to be established . To investigate the effect of PAF on cytokine synthesis, we measured the cytokine concentration in the culture media of two human cell lines: normal endometrial stromal cells (ESC) and endometrial stromal sarcoma cells (MaMi), following stimulation with a non-metabolized PAF analogue, carbamyl-PAF (C-PAF) . Enzyme-linked immunosorbent assays were used to measure five cytokines: interleukin (IL)-6, IL-8, macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein-1alpha (MIP-1alpha) and tumour necrosis factor-alpha (TNF-alpha) . We also evaluated the mRNA expression for IL-6 and IL-8 in ESC after C-PAF stimulation using Northern blot analysis . Non-stimulated ESC and MaMi cells both secreted IL-6, IL-8, and M-CSF, but not MIP-1alpha or TNF-alpha . The concentrations of IL-6, IL-8, M-CSF, MIP-1alpha, and TNF-alpha in the culture media of both cell lines increased in parallel with increasing amounts of C-PAF . C-PAF stimulated IL-6 and IL-8 transcription in ESC . These results suggest that PAF secretion by decidual tissues and developing embryos may induce cytokine synthesis by the ESC, as part of the cytokine network in the feto-maternal unit . An increase in the local cytokine concentration may be an important factor in the maintenance of early stages of gestation.

Ann Plast Surg, 1999 May, 42(5), 496 - 501
Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro; Sagiroglu JS et al.; The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry . Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion . Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal {PF}) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays . The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro . Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay . For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture . The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter . Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant . The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter . Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05) . TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant . Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion.

Cytometry, 1999 May 1, 36(1), 1 - 10
In vitro motility evaluation of aggregated cancer cells by means of automatic image processing; De Hauwer C et al.; BACKGROUND: Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours . METHODS: Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media . Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory . This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated . This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster . RESULTS: The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster . CONCLUSIONS: The proposed equipment enables in vitro aggregated cell motility to be studied . This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds . The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

Eur J Pharmacol, 1999 Apr 16, 370(3), 297 - 305
Esculetin suppresses proteoglycan metabolism by inhibiting the production of matrix metalloproteinases in rabbit chondrocytes; Watanabe K et al.; The possible mechanism of the chondroprotective effect of 6,7-dihydroxycoumarin (esculetin) was investigated using primary cultures of rabbit articular chondrocytes . Esculetin (EST) significantly suppressed the proteoglycan depletion and the release of pulse-labeled {35S}proteoglycan from the matrix layer of rabbit chondrocytes treated with recombinant human interleukin-1alpha . The matrix metalloproteinase inhibitor, 1,10-phenanthroline, also blocked the proteoglycan depletion and {35S}proteoglycan release . From these results, it is likely that recombinant human interleukin-1alpha-induced proteoglycan depletion is mediated by matrix metalloproteinases . Although esculetin did not directly inhibit collagenolytic activity in the culture media, it significantly suppressed the production of pro-matrix metalloproteinase-1/interstitial procollagenase and pro-matrix metalloproteinase-3/prostromelysin 1, accompanied by a decrease in the steady-state levels of their mRNAs . These results suggest that esculetin is a therapeutically effective candidate for inhibition of cartilage destruction in osteoarthritis and rheumatoid arthritis.

Endocr Regul, 1998 Jun, 32(2), 93 - 98
In Vitro Effect of Triiodothyronine on the Cyclic AMP, Progesterone and Testosterone Level in Porcine Theca, Granulosa and Luteal Cells; Gregoraszczuk EL et al.; OBJECTIVE: To investigate the influence of thyroid hormone on steroid production and cAMP accumulation in porcine theca (Tc) and granulosa cells (Gc) isolated from preovulatory follicles as well as in luteal cells isolated during the mid-developing luteal phase . METHODS: Granulosa and theca cells separated from pig ovarian follicles and pieces of corpora lutea were cultured in the incubation medium M199 with 5 % calf serum . After the addition of triiodothyronine (T3) and 3-isobutyl-1-methyl xanthine (IBMX) to the culture medium cAMP, progesterone and testosterone were estimated with the aid of specific RIA . RESULTS: T3 added to the culture media stimulated the steroid secretion and cAMP accumulation in all cell types investigated . In theca cells, T3 alone increased androgen production by 2 fold and the addition of IBMX further augmented the steroidogenesis by 2.2 fold . In granulosa cell culture, IBMX had no effect either on the basal or T3 stimulated progesterone secretion and cAMP accumulation . In luteal cell culture, IBMX added alone increased progesterone secretion and cAMP accumulation in the same manner as T3 . Further augmentation of progesterone secretion (1.3-fold) and cAMP accumulation (1.1-fold) was observed after the addition of IBMX together with T3 . CONCLUSION: The influence of thyroid hormone on cyclic nucleotide release by ovarian cells may suggest the involvement of cAMP-dependent mechanism in the realisation of T3 action in ovarian cells.

Osteoarthritis Cartilage, 1999 May, 7(3), 272 - 80
Anti-interleukin-1 effects of diacerein and rhein in human osteoarthritic synovial tissue and cartilage cultures; Yaron M et al.; OBJECTIVE: The etiology of osteoarthritis (OA) is still a matter of debate . Several factors are known to be involved in the destruction of the articular cartilage . Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors, such as nitric oxide (NO) . The objective of this study was to evaluate the effect of diacerein, a new anti-OA agent and its active metabolite, rhein, on the production and function of IL-1beta, nitric oxide (NO) and receptor agonist (IL-1ra) in human OA cartilage and synovial tissue cultures . DESIGN: Synovial tissue and cartilage derived from OA patients were kept in culture for 48-72 hours in the presence of 1 microg/ml of lipopolysaccharide (LPS) with or without diacerein (10(-7)-10(-5) M), rhein (10(-7)-10(-5) M) and hydrocortisone (5 microg/ml) . IL-1beta, IL-1ra, NO productions and 35S uptake were measured in culture media . In some experiments the resulting supernatants from synovial tissue cultures were added to cartilage . RESULTS: Diacerein and rhein, as well as hydrocortisone, significantly inhibited LPS-induced IL-1beta production by synovial tissue and cartilage . They also significantly reversed the inhibitory effect of LPS on cartilage 35S uptake . Culture media from synovial tissue containing LPS+diacerein (10(-6) M) or +rhein (10(-6) M) had a significantly less inhibitory effect on cartilage synthesis than culture media containing LPS only . Diacerein and rhein decreased NO release in synovial tissue and cartilage media and increased IL-1ra levels in cartilage culture media . CONCLUSION: An inhibitory effect of diacerein and rhein at therapeutic concentrations on both IL-1beta secretion and function in human synovial tissue and cartilage is suggested . Diacerein and rhein effects on NO production by LPS-stimulated OA synovial tissue and cartilage may both contribute and elucidate their anti-OA properties .

Biol Reprod, 1999 Jun, 60(6), 1345 - 52
Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media; Krisher RL et al.; Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids {HECM-6} followed by tissue culture medium 199 + 10% bovine calf serum) . Modifications were made to reduce or eliminate protein . Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured . There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively) . Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%) . There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%) . Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium . Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium . This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.

J Biomed Mater Res, 1999 Jun 15, 45(4), 311 - 21
The influence of hydroxyapatite particles on osteoclast cell activities; Sun JS et al.; Aseptic loosening after total joint arthroplasty is a major problem in orthopedic surgery . Small particles from material wear have been reported as the main cause of implant failure . For this reason, investigation into possible wear particles from the materials used in the implant may lead to longevity after arthroplasty . Hydroxyapatite (HA) has been extensively investigated and reported as an excellent biomaterial with excellent biocompatibility . In this study, we used an in vitro osteoblast/osteoclast model to test the biocompatibility of various-sized HA particles . Primary osteoclasts/osteoblasts were co-cultured with different-sized HA particles (0.5-3.0 microm, 37-53 microm, 177-205 microm, and 420-841 microm) for 3 h, 1 day, 3 days, and 7 days . Cellular responses to the HA particles were evaluated by changes in cell counts and the secretion of transforming growth factor (TGF-beta1), alkaline phosphatase (ALP), tumor necrosis factor (TNF-alpha), prostaglandin (PGE2), and lactate dehydrogenase (LDH) in the supernatant of the culture media . The results showed that osteoblasts/osteoclasts co-cultured with HA particles smaller than 53 microm undergo the most significant changes . Cellular counts significantly decreased, and the changes were more obvious in the osteoblast population . There also was a significant decrease in TGF-beta1 concentration and a significant increase in PGE2 and LDH concentration, but there were no changes in the TNF-alpha or ALP titer . It can be concluded that larger HA particles may be quite compatible with bone cells while smaller-sized HA particles can both activate the osteoclasts and decrease the cell population of the osteoblasts . Justification for the additional expense incurred with the use of hydroxyapatite in primary total hip arthroplasty should be further evaluated.

Prostaglandins Leukot Essent Fatty Acids, 1999 Jan, 60(1), 73 - 6
Regulation of prostaglandin production by nitric oxide in rat smooth muscle myometrial cells; Motta AB et al.; Smooth muscle myometrial cells isolated by an enzymatic method from estrogenized rats were used after 7-10 days of culture . They were incubated for 24 h with two distinct competitive nitric oxide (NO) inhibitors: NG-monomethyl-L-arginine (L-NMMA: 300 microM) and L-nitro-arginine methylester (L-NAME: 600 microM, 5 mM and 10 mM) . Afterwards, the supernatants were separated in order to measure nitrite production and prostaglandin PGE synthesis . In the present report, we demonstrate that myometrial cells from estrogenized rats are able to produce NO, since all the inhibitors significantly decrease the production of nitrites in the culture media . Furthermore, we report that both inhibitors inhibited PGE synthesis by myometrial cells . We also used a donor of NO in the incubation medium for 24 h, sodium nitroprusside (NP), obtaining an strong (P< 0.001) increase in both nitrite and PGE production . We conclude that myometrial cells can produce NO and that one possible role of the NO synthetized by this cells may be the modulation of PGE production.

Proc Natl Acad Sci U S A, 1999 May 11, 96(10), 5545 - 8
Patterning cells and their environments using multiple laminar fluid flows in capillary networks; Takayama S et al.; This paper describes the use of laminar flow of liquids in capillary systems to pattern the cell culture substrate, to perform patterned cell deposition, and to pattern the cell culture media . We demonstrate the patterning of the cell culture substrate with different proteins, the patterning of different types of cells adjacent to each other, the patterned delivery of chemicals to adhered cells, and performing enzymatic reactions over select cells or over a portion of a cell . This method offers a way to simultaneously control the characteristics of the surface to which cells are attached, the type of cells that are in their vicinity, and the kind of media that cells or part of a cell are exposed to . The method is experimentally simple, highly adaptable, and requires no special equipment except for an elastomeric relief that can be readily prepared by rapid prototyping.

Invest Ophthalmol Vis Sci, 1999 May, 40(6), 1285 - 8
Pressure-induced syneretic response in rhesus monkey lenses; Bettelheim FA et al.; PURPOSE: To investigate the effect of pressure on the freezable and nonfreezable water content of the lens . METHODS: Excised rhesus monkey lenses in tissue culture media were subjected to three different hydrostatic pressures (2 atm, 1 atm, and 0.03 atm) for 24 hours . Then while still under the experimental pressure, the vessels were cooled in dry ice-acetone until the lenses were frozen . While the lenses were kept frozen, nuclear and cortical samples were dissected, enclosed in a sample pan, and weighed . Differential scanning calorimetry (DSC) measurements were performed between -30 degrees C and 30 degrees C . Total water content of each lens sample was obtained by thermogravimetric analysis at 105 degrees C . The nonfreezable water content was obtained by subtracting the freezable water content calculated from the DSC data from the total water content . RESULTS: The total water content of the lenses did not change significantly as a function of pressure applied . This was true both for cortical and for nuclear sections . The freezable water content increased as the pressure decreased both in cortex and nucleus . Similarly, the freezable water/nonfreezable water ratio also decreased with increasing pressure . CONCLUSIONS: External hydrostatic pressure would generate an influx of water into the lens . To alleviate this diluting tendency and to prevent turbidity as a result of dilution, the lens must effect an osmotic pressure change equivalent to the applied pressure . Change in the osmotic pressure is caused by changing the activity of the water (i.e., converting free water to bound water) . This is a reversible and energetically the least expensive response . The release of bound water from the hydration layers of macromolecules and its conversion to free water in condensed systems are known as syneresis . In the lens decreasing pressures induce syneresis as demonstrated by the increase in freezable water content and the freezable water/nonfreezable water ratio . Such a response may be operative also in accommodating lenses.

FEMS Microbiol Lett, 1999 May 1, 174(1), 49 - 55
An extracellular acid stress-sensing protein needed for acid tolerance induction in Escherichia coli; Rowbury RJ et al.; An extracellular induction component (EIC), needed for acid tolerance induction at pH 5.0 in Escherichia coli, arises from an extracellular precursor which senses acid stress and is activated (forming the EIC) by such stress . The precursor, which is a heat-stable protein, was formed by cells which had not been subjected to acid stress, being present in culture media after growth at pH values from 7.0 to 9.0 . This stress-sensing molecule was activated to the EIC at pH values from 4.5 to 6.0 but not at pH 6.5 and did not form EIC on incubation at an extremely acidic pH e.g . 2.0 . The precursor was not inactivated at pH 2.0 . Precursor activation might be reversible, as the EIC lost its ability to induce acid tolerance after incubation at pH 9.0, but regained it if subsequently incubated at pH 5.0 . Whereas the sensor formed at pH 7.0 can only be activated at pH 5.0 to 6.0, that synthesized at pH 9.0 can be activated at pH 5.0 to 7.5 . Accordingly, this work shows that the acid stress sensor is extracellular, and it is proposed that its presence in the medium rather than in the cells, allows more sensitive and rapid responses to acid stress.

Br J Clin Pharmacol, 1999 Apr, 47(4), 433 - 9
Effect of troglitazone on plasma lipid metabolism and lipoprotein lipase; Kobayashi J et al.; AIMS: To clarify how troglitazone, an insulin-sensitizing agent, affects lipid metabolism and postheparin plasma lipoprotein lipase (LPL) . METHODS: Fifteen patients (3 male, 12 female) (the average age 62+/-7 years; the mean body mass index (BMI) 25+/-3 kg/m2 ) were recruited for this study . The serum lipids and postheparin plasma lipoprotein lipase (LPL) mass before and 4 weeks after oral administration of troglitazone (200 mg day-1 ) were measured . A mouse preadipocyte cell line, 3T3-L1, was incubated with troglitazone and LPL enzyme protein mass in the culture media was measured by an enzyme linked immunosorbent assay . A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for the carboxyl terminal 135 amino acid of mouse LPL cDNA was used to evaluate the effect of troglitazone on expression of LPL and Northern blot analysis carried out to determine expression of LPL . RESULTS: The average levels before treatment of fasting serum total cholesterol, triglycerides, high density lipoprotein cholesterol, plasma glucose and glycohaemoglobin A1c were 5.6+/-0.9, 1.8+/-1.0, 1.5+/-0.5, 8.1+/-1.7 mmol l-1 and 7.8+/-1.6% respectively . Four weeks after treatment, those levels were 5.4+/-0.9, 1.2+/-0.3 (P=0.004), 1.6+/-0.5 (P=0.02) mmol l-1, 7.7+/-2.3 mmol l-1 and 7 . 3+/-0.6% (P=0.01), respectively . The postheparin plasma LPL mass increased from 226+/-39 to 257+/-68 ng ml-1 (P=0.03) during that period . The LPL mass in the media of 3T3 L1 cells cultured in the presence of 10, 20 or 30 microm of this compound increased in a dose dependent manner . RT-PCR revealed that the area of the bands of the RT-PCR products on 1.5% agarose gel analyzed with NIH image from the cell extracts cultured in the presence of 10 microm troglitazone was significantly larger (P=0.0069) than that in the absence of this compound . Northern blot analysis revealed that in the cultured 3T3-L1 cells, the expression of LPL was enhanced in the presence of 10 microm troglitazone . CONCLUSIONS: Troglitazone improves plasma triglyceride-rich lipoproteins metabolism by enhancing the expression of LPL in adipocytes.

Prep Biochem Biotechnol, 1999 May, 29(2), 189 - 202
Continuous hydrolysis of goat whey in an ultrafiltration reactor: generation of alpha-lactorphin; Bordenave S et al.; Bovine whey hydrolysate has been developed and applied to areas such as nutrition, culture media, and isolation of bioactive peptides . In order to produce such a type of hydrolysate, it is possible to use goat whey, which constitutes also a food processing by-product . Enzymatic hydrolysis of goat whey by pepsin was carried out in a continuous ultrafiltration reactor . The permeate contained peptide hydrolysate that was resolved by RPHPLC . Second order derivative spectroscopy, amino acid analysis, and mass spectrometry revealed the presence of a biologically active peptide called alpha-lactorphin . This constitutes preliminary information about goat whey enzymatic degradation for future applications.

Pancreas, 1999 May, 18(4), 403 - 9
Dissociated insulin and islet amyloid polypeptide secretion from isolated rat pancreatic islets cocultured with human pancreatic adenocarcinoma cells; Wang F et al.; Abnormal insulin and islet amyloid polypeptide (IAPP) secretion are usually seen in patients with exocrine pancreatic cancer . The beta-cell dysfunction is a characteristic of the glucose intolerance found in pancreatic cancer patients . The effects of pancreatic cancer cells on insulin and IAPP secretion from beta cells are unclear . In this study, isolated rat pancreatic islets were cocultured with two human pancreatic adenocarcinoma cell lines (Panc-1 and HPAF) and a human colonic adenocarcinoma cell line (HT-29) . As a control, islets were incubated in the absence of malignant cells . The accumulation of insulin and IAPP in culture media was measured by radioimmunoassay . Output of insulin and IAPP was decreased in islets cocultured with each malignant cell line . Molar ratio of secreted IAPP and insulin (IAPP/insulin) was increased in the islets cocultured with Panc-1 or HPAF cells, but not HT-29 cells . The decreased insulin and IAPP secretion were partly recovered after Panc-1, HPAF, or HT-29 cells were removed . The IAPP/insulin ratio was normalized after the removal of Panc-1 or HPAF cells . This study indicates that insulin and IAPP secretion are altered by the human adenocarcinoma cells investigated . The impairment induced by pancreatic adenocarcinoma cells is associated with a hypersecretion of IAPP relative to insulin on a molar basis.

J Vasc Surg, 1999 May, 29(5), 884 - 92; discussion 892-3
Indomethacin inhibits expansion of experimental aortic aneurysms via inhibition of the cox2 isoform of cyclooxygenase; Miralles M et al.; PURPOSE: Cyclooxygenase, either the cox1 or cox2 isoform, controls synthesis of prostaglandin E2 (PGE2), which regulates expression of matrix metalloprotease-9 (MMP-9) . PGE2 and MMP-9 are elevated in aortic aneurysms . The mechanisms and time course of the inhibition of aneurysm expansion with a nonspecific cyclooxygenase inhibitor, indomethacin, were determined in an animal model . METHODS: Rats underwent aortic perfusion with saline (n = 40) as controls or with elastase . Elastase-treated animals received no treatment (n = 82) or received indomethacin (n = 73) . Aortic diameters were determined at the time of aortic perfusion and when the rats were killed . The aortas were harvested and used for whole organ culture, substrate gel zymography, or histologic analysis . RESULTS: The control group demonstrated little change in aortic diameter . All the elastase-only animals developed aneurysms (maximal aortic diameter, 5.27 +/- 2.37 mm on day 14) . Indomethacin markedly decreased the rate of aortic expansion (maximum aortic diameter, 3.45 +/- 1.11 mm; P <.001 vs the elastase-only group) . The enzyme-linked immunosorbent assay of aortic explant culture media showed that PGE2 synthesis paralleled aortic expansion, and indomethacin decreased PGE2 synthesis . Histologically, the aortic elastin architecture was destroyed in the elastase group, but was preserved with indomethacin treatment . In situ, hybridization for cox1 and cox2 showed that cox2, but not cox1, was expressed and was co-localized by immunohistochemistry to macrophages associated with the aortic wall . Decreased levels of MMP-9 activity with indomethacin were shown by means of substrate zymography . MMP-9 was also localized to macrophages . CONCLUSION: Indomethacin attenuates aneurysm growth, and its effects are mediated via inhibition of the cox2 isoform of cyclooxygenase, which decreases PGE2 and MMP-9 synthesis.

Bioelectrochem Bioenerg, 1999 Feb, 48(1), 237 - 41
Surface charge density and light-scattering of the Plectonema boryanum spheroplasts; Doltchinkova V; The electrokinetic properties of spheroplasts from the cyanobacterium Plectonema boryanum were examined by particle microelectrophoresis technique . The electrophoretic mobility (EPM) of the particles was determined after incubation with CaCl2 in dependence of iron content in culture media as follows: an iron sufficient medium ('control' variant), an iron-deficient medium ('Fe-starved' variant) and an excess of iron supply medium ('20 x Fe' variant) . Strong increase in EPM was observed with micromolar concentrations of divalent cations at '20 x Fe' spheroplasts . This pattern of calcium efficiency was not accompanied with the cation influences on the aggregate ability of particles . The EPM of 'control' spheroplasts strongly decreased with addition of calcium cations . The 'Fe-starved' spheroplasts were characterized with a slight reduction in EPM and a mild change in light-scattering properties of the particles . The data is the direct demonstration of the interaction between calcium cations and spheroplast surface, which could be proposed to play a role in the environmental cycling of iron.

Lancet, 1999 Apr 24, 353(9162), 1401 - 3
Effect of fly control on trachoma and diarrhoea; Emerson PM et al.; BACKGROUND: Domestic flies are accepted vectors of diarrhoea, but their role in trachoma transmission has never been quantified and no study has shown that fly control decreases the prevalence of trachoma . We assessed the effect of fly control on public health in a pilot study in Gambian villages . METHODS: We studied two pairs of villages--one pair in the 1997 wet season, and one pair in the 1998 dry season . For each pair, deltamethrin was sprayed for 3 months to control flies in one village whilst the other was used as a control . Fly populations were monitored with traps . We surveyed trachoma at baseline and at 3 months, and collected daily data on diarrhoea in children aged between 3 months and 5 years . FINDINGS: Fly control decreased numbers of muscid flies by around 75% in the intervention villages compared with controls . Trachoma prevalence was similar at baseline (wet season, prevalence in intervention village 8.8% vs control 12.2%; dry season, 18.0% vs 16.0%), but after 3 months of fly control there were 75% fewer new cases of trachoma in the intervention villages (wet season 3.7% vs 13.7%; dry season 10.0% vs 18.9%; rate ratio and relative risk of pooled data 0.25 {adjusted 95% CI 0.09-0.64}, p=0.003) . There was 22% less childhood diarrhoea in the wet season (14% vs 19%, period prevalence ratio 0.78 {0.64-0.95}, p=0.01), and 26% less diarrhoea in the dry season (6% vs 8%; 0.74 {0.34-1.59}, p=0.60) compared with controls . INTERPRETATION: Muscid flies are important vectors of trachoma and childhood diarrhoea in The Gambia . Deltamethrin spray is effective for fly control and may be useful for reducing trachoma and diarrhoea in some situations, but further research on sustainable fly-control methods is neededPIP: The causative agent of trachoma, Chlamydia trachomatis, has been found on flies fed on heavily infected laboratory culture media . Findings are presented from an assessment of the effect of domestic fly control upon the prevalence of trachoma and associated cases of childhood diarrhea in 2 pairs of Gambian villages . 1 pair of villages was studied in the 1997 wet season and the second pair in the 1998 dry season . For each pair, deltamethrin was sprayed for 3 months to control flies in 1 village, while the other village was used as a control . Fly populations were monitored with traps . The prevalence of trachoma was measured at baseline and at 3 months, and data were collected daily on diarrhea in children aged 3 months to 5 years . Fly control decreased the numbers of muscid flies by approximately 75% in the intervention villages compared with controls . While the prevalence of trachoma was similar at baseline between study and control villages, after 3 months of fly control there were 75% fewer new cases of trachoma in the intervention villages . There was 22% and 26% less childhood diarrhea in the wet and dry seasons, respectively, compared with controls . These findings demonstrate that muscid flies are important vectors of trachoma and childhood diarrhea in The Gambia, and that the use of deltamethrin spray can help to reduce the prevalence of both trachoma and associated diarrhea cases .

J Clin Invest, 1999 May, 103(9), 1345 - 52
IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis; Kotake S et al.; IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated . Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices . Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2) . Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells . In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts . ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts . Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures . Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients . Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues . These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

Curr Eye Res, 1999 Feb, 18(2), 130 - 4
Serum-free cultivation of human Tenon's capsule fibroblasts; Denk PO et al.; PURPOSE: In the present study, the effect of three different serum-free and one serum-containing control medium on adhesion, proliferation, cryopreservation and PDGF-induced effects on cell proliferation of human Tenon's capsule fibroblasts (HTF) was compared . MATERIALS AND METHODS: Third passage HTF were suspended in four different culture media (WM/F12, WM/F12/FCS 1%, LR-1, DMEM) and plating efficiency was determined after 24h using a cell counter system . Subsequently, cells were seeded at a density of 50/mm2 and cultured for ten days using the different culture media . Cell number was determined at day 2, 4, 7 and 10 after seeding . Furthermore, HTF cultured under the different conditions were stimulated by PDGF-BB {50 ng/ml} . Additionally, cell vitality after two weeks cryopreservation in five different culture media (WM/F12, WM/F12/FCS 1%, WM/F12/FCS 20%, LR-1, DMEM) was determined . RESULTS: The plating efficiency of HTF when seeded in serum-free medium ranged from 55.3% to 59.6% . Using serum containing WM/F12/FCS 1% a slightly higher plating efficiency of 74.8% was obtained . Proliferation assays revealed population doublings of 0.77 with WM/F12/FCS 1% after an incubation period of 10 days . Cultivation of HTF using serum-free conditions did not cause significant cell proliferation but a slight cell loss which ranged from 23.1% to 34% . Addition of PDGF-BB resulted in a significant increase in cell proliferation with WM/F12/FCS 1%, WM/F12 and DMEM . After two weeks of cryopreservation in WM/F12, LR-1, DMEM, WM/F12/FCS 1% and WM/F12/FCS 20%, only the application of high serum concentrations led to sufficient preservation of cell vitality with a plating efficiency of 82.9% . DISCUSSION: The results of the present study demonstrate that the use of serum-containing media is mandatory for cryopreservation of HTF . Seeding of cells can be performed either with serum or without serum . HTF cultured under serum-free conditions can be maintained quiescent with a sufficient number of cells remaining vital . The serum-free media used in this study can be applied for the investigation of cytokine effects on HTF.

Hum Reprod, 1999 Mar, 14(3), 774 - 81
Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture; Fong CY et al.; Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts . A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2 . Each patient's day 2 embryos were then equally allotted to two in-vitro systems . Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil . The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only . The same culture protocol was used for system B without Vero cells . Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere . Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis . Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4 . The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems . TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages . There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01) . The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos.

Free Radic Biol Med, 1999 Mar, 26(5-6), 679 - 84
Helicobacter pylori-associated gastric pro- and antioxidant formation in Mongolian gerbils; Suzuki H et al.; Helicobacter pylori colonized gastric mucosa is manifest in a significant neutrophil infiltration with an extensive level of oxyradical formation . Mongolian gerbil is one of the excellent models for H . pylori-infection . The present study was designed to investigate pro- and antioxidant formation in the stomach of H . pylori-positive gerbils . Fourteen male Mongolian gerbils (MGS/Sea) were orally inoculated with H . pylori (ATCC43504) (Hp group) and 15 gerbils were inoculated with the culture media (Control) . H . pylori infection was confirmed by the serum anti-H . pylori IgG test . Each gerbil was evaluated 6 or 12 weeks after the inoculation . Neutrophil infiltration was assessed by the tissue MPO activity . Mucosal oxidative stress was evaluated by thiobarbituric acid-reactive substances (TBARS), total glutathione contents, glutathione peroxidase (GSHPx) activity and Cu-, Zn-superoxide dismutase (SOD) activity . In Hp group, the H . pylori was persistently infected until 12 weeks . The level of MPO activity was significantly higher in Hp group at 6 and 12 weeks . Although the levels of TBARS and total glutathione were within the same range as controls at 6 weeks, they were significantly increased at 12 weeks . However, GSHPx activity was significantly increased at 6 weeks, but became the same range with the controls at 12 weeks . SOD activity showed no significant increase in Hp group at 6 and 12 weeks . In conclusion, H . pylori inoculation induced gastric mucosal neutrophil activation and pro-oxidant formation and also increased total glutathione contents, one of the mucosal antioxidants in gerbils.

Electrophoresis, 1999 Mar, 20(3), 626 - 9
Two-dimensional electrophoresis of Cereus peruvianus (Cactaceae) callus tissue proteins; Mangolin CA et al.; Two-dimensional electrophoresis of Cereus peruvianus callus tissues grown in culture media containing two different 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin combinations was used to identify minor differences in polypeptide composition of these cell clones . Altered expression during growth in the two 2,4-D and kinetin combinations was apparent for 13 polypeptides when calluses in the two media were compared . The number of proteins with differential expression (presence or absence of specific spots) was higher in callus tissues cultured in the 4.0 mg/L 2,4-D and 8.0 mg/L kinetin combination than in callus tissues cultured in the 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination . The present results show that the callus tissues maintained at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin can be used as a matrix for in vitro selection programs.

Biochim Biophys Acta, 1999 Apr 19, 1427(2), 265 - 75
Considerations for in vitro retinoid experiments: importance of protein interaction; Klaassen I et al.; Retinoids, natural and synthetic substances structurally related to vitamin A, are important modulators of cell proliferation and differentiation, and have proven activity in cancer therapy . Experiments to reveal the mechanism of action of retinoids are routinely performed in in vitro models . As retinoids are relatively hydrophobic and unstable, we hypothesized that the composition of culture media is of critical importance for the stability and bioavailability of these compounds . Various culture media were incubated with all-trans-, 13-cis- and 9-cis-retinoic acid (RA) . Without fetal calf serum (FCS) or bovine serum albumin (BSA) in the medium, the concentration of these retinoids was found to decrease to considerably low levels . This excessive loss of retinoids was due to absorption to culture plates, reaction tubes and pipet tips . Binding of retinoids to BSA was demonstrated to have attenuating effects on uptake and metabolism of all-trans-RA, as studied in oral keratinocytes and head and neck cancer cells, indicating that a balance exists between the bioavailability and the aspecific loss of retinoids . In this study we demonstrate that the type of culture medium and especially the presence of protein in the medium is of paramount importance to perform reproducible experiments with retinoids.

Biochem J, 1999 May 1, 339 ( Pt 3), 571 - 7
Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism; Sztrolovics R et al.; A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period . An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium . This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase . Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation . By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period . In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period . Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period . Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter . These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan . This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration.

Biol Reprod, 1999 May, 60(5), 1183 - 7
Expression of hepatocyte growth factor in cultured human endometrial stromal cells is induced through a protein kinase C-dependent pathway; Nasu K et al.; To examine the production of hepatocyte growth factor (HGF) by human endometrial stromal cells (ESC) in vitro, concentrations of HGF in the culture media of ESC were measured after the addition of various amounts of 12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin, lipopolysaccharide (LPS), interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha (TNFalpha), interferon-gamma (IFNgamma), or ethynylestradiol-17alpha using an ELISA . The expression of HGF mRNA was also assayed by a reverse transcription-polymerase chain reaction . The concentration of HGF in the culture media of unstimulated ESC was below the detection level of the assay . TPA stimulated the secretion of HGF by ESC in a dose-dependent manner . TPA also induced the transcription of HGF mRNA by ESC . Forskolin, LPS, IL-1beta, IL-6, IL-8, TNFalpha, IFNgamma, or ethynylestradiol-17alpha did not alter HGF mRNA or protein levels . TPA-stimulated production of HGF was partially inhibited by the addition of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine or sphingosine . These results suggest that a protein kinase C-dependent pathway may play an important role in the regulation of HGF production by ESC . HGF secreted by ESC may be involved in the regeneration of the endometrium during the normal menstrual cycle.

J Periodontal Res, 1999 Feb, 34(2), 97 - 104
Prostaglandin E2 and I2 regulate intercellular adhesion molecule-1 expression in interleukin-1 beta-stimulated human gingival fibroblasts; Iwasaki K et al.; The present study investigated the effect of prostaglandin (PG) E2 and PGI2 on intercellular adhesion molecule-1 (ICAM-1) expression in interleukin-1 beta (IL-1 beta)-stimulated human gingival fibroblasts (HGF) . IL-1 beta potently induced ICAM-1 expression in HGF and indomethacin, a cyclooxygenase inhibitor, enhanced ICAM-1 expression in the cells . These data showed that endogenous PGs generated by HGF stimulated with IL-1 beta downregulated ICAM-1 expression . IL-1 beta significantly increased the levels of PGE2 and, to a lesser extent, those of 6-keto-PGF1 alpha (a stable metabolite of PGI2) in the culture media of HGF . Indomethacin completely inhibited the production of PGE2 and 6-keto-PGF1 alpha in IL-1 beta-stimulated HGF . Exogenous PGE2 and carbacyclin (a stable derivative of PGI2) in the presence of indomethacin dose-dependently suppressed ICAM-1 expression in IL-1 beta-challenged HGF . Since PGE2 and PGI2 are known to elevate intracellular cyclic AMP (cAMP) levels, we examined the effect of dibutyryl cAMP, a cAMP analogue, and isobutylmethylxanthine, a phosphodiesterase inhibitor, on ICAM-1 expression . Both agents downregulated ICAM-1 expression in IL-1 beta-stimulated HGF . These results suggest that PGE2 and PGI2 downregulate ICAM-1 expression in IL-1 beta-stimulated HGF through a cAMP-dependent mechanism and that intracellular cAMP elevation in HGF may control inflammatory and immune responses in periodontal disease.

J Hepatol, 1999 Apr, 30(4), 612 - 20
Transformation-dependent calcium influx by voltage-operated calcium channels in stellate cells of rat liver; Roth-Eichhorn S et al.; BACKGROUND/AIMS: The transformation of hepatic stellate cells into myofibroblasts is a key step in the pathogenesis of fibrotic liver diseases . The intracellular signaling associated with hepatic stellate cell transformation becomes a point of interest, especially the role of cytosolic free calcium concentration ({Ca2+}i) . The aim of the study was to investigate possible differences between various transformation phenotypes of hepatic stellate cells with regard to the calcium influx mediated by L-type voltage-operated calcium channels (L-type VOC) . METHODS: Hepatic stellate cells were isolated from rat liver by pronase-collagenase reperfusion and cultured under standard conditions . The transformation of hepatic stellate cells was stimulated by treatment with transforming growth factor-beta (TGF-beta) or inhibited with interferon-gamma (IFN-gamma) and characterized by immunocytochemistry for smooth muscle alpha-actin and determination of hyaluronan in the culture media with a ligand binding assay . {Ca2+}i was measured in individual cells with fluorescence microscopy using fura-2 . VOCs were activated by the standard procedure of extracellular potassium elevation, to achieve depolarization, and identified by various controls . RESULTS: In transformed myofibroblasts the activation of VOCs by potassium elevation from 5.4 mmol/l to 50.4 mmol/l led to a 19% increase in {Ca2+}i in contrast to 0.2% in hepatic stellate cells cultured for 3 days . In 7-day old hepatic stellate cells, after stimulation of cell transformation with TGF-beta-1, an enhanced {Ca2+}i response to potassium elevation was detected, while inhibition of transformation with IFN-gamma for the same time caused a decreased calcium signal compared with untreated control cultures . Short-term treatment with the cytokines (1 day) did not influence depolarization-dependent calcium signals . CONCLUSION: The results show the {Ca2+}i increase via L-type VOCs to be dependent on the transformation level of hepatic stellate cells into myofibroblasts which can be influenced by the long-term treatment of hepatic stellate cells with TGF-beta or IFN-gamma . In contrast, there is no evidence for direct regulation of VOC activity by TGF-beta or IFN-gamma after short-term exposure.

J Gastroenterol, 1999 Feb, 34(1), 66 - 74
Mucosal proinflammatory cytokine production correlates with endoscopic activity of ulcerative colitis; Ishiguro Y; Proinflammatory cytokines are believed to be involved in the pathogenesis of ulcerative colitis (UC) . The aim of this study was to clarify the profiles of proinflammatory cytokine production in patients with UC in terms of disease intractability, endoscopic findings, and host response to lipopolysaccharide (LPS) stimulation . Colonic mucosal tissues were obtained from patients with active UC (n = 15, including 4 patients with intractable disease) and inactive UC (n = 7), non-inflammatory bowel disease (IBD) colitis (n = 11), and controls (n = 20) . Organ culture was performed, and the amounts of four cytokines (described below) in the culture media were determined by enzyme-linked immunosorbent assay (ELISA) . LPS stimulation enhanced interleukin (IL)-1beta, IL-8, and IL-6 production in colonic specimens from all groups, but enhanced tumor necrosis factor (TNF)-alpha production only in active UC specimens . Levels of IL-6, IL-8, and TNF-alpha were significantly higher in active UC than in non-IBD colitis, and the production of all three of these cytokines was correlated to the endoscopic grade of inflammation . The production of these cytokines was also significantly higher in patients with intractable disease receiving corticosteroids than in patients with non-intractable disease receiving corticosteroids . These results suggest that enhanced production of mucosal proinflammatory cytokines may be implicated in the pathogenesis of UC.

Calcif Tissue Int, 1999 May, 64(5), 402 - 13
Interleukin-1beta-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro; Chien HH et al.; The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis . PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics . Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1 . PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics . In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules . Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals . The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches . Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group . Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group . Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture media after {35S}-methionine labeling showed their deposition primarily in the mineralized nodules of the Dex group, and their release into the media in the IL-1 group . Immunogold labeling demonstrated the location of OPN and BSP in mineralized nodules of the Dex group, but no significant labeling occurred in the nodule-like structures from the IL-1 group . Interestingly, IL-1 treatment increased the expression of collagenase mRNA by sevenfold, compared with that of the Dex group . These data suggest that the IL-1-induced formation of unmineralized nodules by PDL cells results not so much from the downregulated formation of matrix proteins, which plays a crucial role in the mineralization process, as from their release into the culture media . Finally, collagenase synthesis upregulated by IL-1 may be involved in this process.

J Biotechnol, 1999 Mar 26, 69(1), 39 - 45
Parameters influencing human mu opioid receptor over-expression in baculovirus-infected insect cells; Massotte D et al.; The cDNA encoding the human mu opioid receptor (hMOR) was cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter . We investigated the influence of different molecular constructions on receptor expression levels: the receptor was fused either to an amino- or a carboxy-terminal histidine tag (hMOR-N-His and hMOR-C-His respectively), or to the cleavable sequence signal of the baculovirus gp64 glycoprotein (gp-hMOR and gp-hMOR-C-His) . Two cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (BTI-TN-5B1-4), in combination with three different culture media were also tested for their ability to produce maximal protein expression . Molecular constructions and culture conditions were both shown to influence substantially protein production . The best results were obtained using cells adapted to serum-free medium combined with constructions in fusion with the endogenous signal sequence of the baculovirus gp64 protein . Those conditions led to maximal expression and shortened the time required for receptor production . We also showed that an amino-terminal location of a hexahistidine tag was more detrimental to the expression level than a carboxy-terminal position.

Kidney Int, 1999 Apr, 55(4), 1259 - 67
Hypoxia-induced adrenomedullin production in the kidney; Nagata D et al.; BACKGROUND: Adrenomedullin (AM) is a newly discovered peptide that has a potent vasorelaxant activity . To investigate its potential roles in hypoxia-induced renal injury, we examined whether AM production in the kidney increased under hypoxic conditions . METHODS: The AM transcript levels in Madin-Darby canine kidney (MDCK) cells, rat vascular smooth muscle cells (VSMCs), and rat mesangial cells were assessed by Northern blot analyses under normoxic and hypoxic conditions . The AM peptide in culture media was measured by radioimmunoassay . The effects of hypoxia on accumulation of cAMP in VSMCs were also examined . The stability of AM transcripts under normoxic and hypoxic conditions was compared in the presence of actinomycin D . The effects of hypoxia on AM promoter activity was assessed by transient transfection assays using the AM promoter subcloned upstream of luciferase gene . RESULTS: The expression of AM transcripts increased significantly in MDCK cells, rat VSMCs, and rat mesangial cells under hypoxic conditions without changes in the stability of AM transcripts; however, the AM promoter activity under hypoxic was not elevated significantly . The accumulation of AM peptide in culture media also increased significantly under hypoxic conditions in MDCK cells (2.2 +/- 0.1 fmol/10(5) cells in normoxia vs . 3.5 +/- 0.3 fmol/10(5) cells in hypoxia, 6 hr after hypoxia induction, P < 0.001), and in rat VSMCs (5.5 +/- 0.3 fmol/10(5) cells in normoxia vs . 7.8 +/- 0.4 fmol/10(5) cells in hypoxia, 8 hr after hypoxia induction, P < 0.01) . Under hypoxic conditions, cAMP levels in rat VSMCs increased significantly compared with those under normoxic conditions (13.3 +/- 1.4 pmol/well vs . 4.6 +/- 0.4 pmol/well, P < 0.01) . CONCLUSIONS: Renal parenchymal cells as well as renal vessels may produce AM under hypoxic conditions.

J Clin Endocrinol Metab, 1999 Apr, 84(4), 1420 - 3
Urocortin stimulates placental adrenocorticotropin and prostaglandin release and myometrial contractility in vitro; Petraglia F et al.; Urocortin is a new member of the CRF family . Multiple biological effects for urocortin have been shown in rats and in some in vitro models, showing a modulatory role in hormonal and behavioral functions . Human placenta expresses urocortin, but no information is available on the possible local biological actions . The aim of the present study was to evaluate the effect of urocortin on placental ACTH and prostaglandin (PG) secretion, as well as on myometrial contractility . Various in vitro models were used . For investigating the effect of urocortin on ACTH release, primary cultures of human trophoblast cells were used . Culture media, collected before and after 3 h exposure to different doses of urocortin and ACTH, were measured by RIA . Trophoblast tissue explants were incubated for 24 h in the presence of increasing doses of urocortin, and prostaglandin E2 (PGE2) levels were measured by RIA . Strips of myometrial tissue were incubated in an organ bath and connected to an isometric smooth-muscle transducer in the presence of urocortin, with or without prostaglandin F2alpha (PGF2a) . In all these experiments, the effect of astressin (a CRF receptor antagonist) on urocortin-induced actions and the effect of equimolar doses of CRF were evaluated . A dose-related increase of trophoblast ACTH or PGE2 was induced by urocortin, whereas astressin inhibited urocortin-stimulated ACTH or PGE2 release . Equimolar doses of CRF showed a similar effect on both ACTH and PGE2 . Urocortin increased PGF2alpha-induced myometrial contractility, and this effect was completely abolished by the addition of astressin . The present study showed that human urocortin stimulates placental secretion of ACTH and PGE2, and modulates myometrial contractility, suggesting a role for this peptide in placental and intrauterine CRF pathways.

J Hematother, 1999 Feb, 8(1), 63 - 73
In vitro culture of acute myelogenous leukemia blasts: a comparison of four different culture media; Bruserud O et al.; Proliferative responses and cytokine secretion were compared when AML blasts were cultured in the three serum-free media, X-Vivo 10, X-Vivo 15, and defined serum-free medium (IMDM with mercaptoethanol, low-density lipoprotein, albumin, and transferrin) and in media containing 10% inactivated fetal bovine serum (FBS) . The following AML blast functions were investigated: (a) constitutive cytokine secretion, (b) autonomous and cytokine-dependent proliferation, and (c) accessory cell function during T cell activation . Constitutive cytokine secretion and accessory cell function differed markedly when using different culture media . For the constitutive AML blast secretion of IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, no qualitative differences were seen, but quantitative differences were observed with decreased cytokine levels for cells cultured in X-Vivo 10 and X-Vivo 15 . The accessory cell function of AML blasts was also decreased in the X-Vivo media, whereas differences were less pronounced when comparing AML blast proliferation . Our results clearly demonstrate that a well-characterized culture system is essential for in vitro studies of AML blast functions.

Biochem J, 1999 Apr 15, 339 ( Pt 2), 233 - 9
Glucose and thyroid hormone co-regulate the expression of the intestinal fructose transporter GLUT5; Matosin-Matekalo M et al.; Expression of the fructose transporter GLUT5 in Caco-2 cells is controlled by the carbohydrate content of the culture media {Mesonero, Matosin, Cambier, Rodriguez-Yoldi and Brot-Laroche (1995) Biochem . J . 312, 757-762} and by the metabolic status of the cells {Mahraoui, Takeda, Mesonero, Chantret, Dussaulx, Bell, and Brot-Laroche (1994) Biochem . J . 301, 169-175} . In this study we show that, in fully differentiated Caco-2/TC7 cells, thyroid hormone and glucose increase GLUT5 mRNA abundance in a dose-dependent manner . Using Caco-2/TC7 cells stably transformed with various fragments of the GLUT5 promoter inserted upstream of the luciferase reporter gene, we localized the sequences that confer 3,3',5-l-tri-iodothyronine (T3)- and/or glucose-sensitivity to the gene . Glucose responsiveness is conferred by the -272/+41 fragment of the promoter, but it is only with the -338/+41 region that transcription of the luciferase reporter gene is stimulated by T3 . This 70 bp fragment from position -338 to -272 of the GLUT5 gene is able to confer T3/glucose-responsiveness to the heterologous thymidine kinase promoter . Electrophoretic-mobility-shift assays demonstrate that thyroid hormone receptors alpha and beta are expressed in Caco-2/TC7 cells . They further show that the -308/-290 region of the GLUT5 promoter binds thyroid hormone receptor/retinoid X receptor heterodimers, and that glucose and/or T3 exert a deleterious effect on the binding of the nuclear protein complex.

Am J Vet Res, 1999 Mar, 60(3), 363 - 7
Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture; Ellington JE et al.; OBJECTIVE: To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA . SAMPLE POPULATION: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase . PROCEDURE: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA . Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures . Spermatozoal survival and motility characteristics were observed over time for all culture methods . Number of spermatozoa attaching to OEC were compared for cocultures . RESULTS: Use of TM without BSA altered spermatozoal function in cell-free medium culture and OEC coculture . A higher percentage of spermatozoa were acrosome reacted in TM with BSA, although percentages of capacitated spermatozoa did not differ . Spermatozoa survived longer and maintained superior motion in TM culture without BSA and in OEC cocultures . More spermatozoa were able to attach to OEC in TM without BSA . CONCLUSIONS: Incubation of cryopreserved spermatozoa in media with BSA resulted in rapid decrease in percentage of intact, motile spermatozoa and limited their ability to interact with OEC . CLINICAL RELEVANCE: Current culture media used for assisted reproduction techniques in horses do not provide functionally capacitated spermatozoa . Removal of BSA from such media improves spermatozoal quality and survival.

What Is Bioreactor?, What Is Growth Medium?, What Is Nitrification?, What Is Environmental Microbiology?, What Is Botulism?, i, Microorganism, e, Microbe, i, Microbes, a, Bacterium, i, Bacteria, r, Escherichia coli, r, Klebsiella, i, Escherichia coli, r, Vibriosis, o, Bacillus, n, S. cerevisiae, r, Penicillin, e, Yeasts, n, Bacteriological, o, Fermentations, a, Leuconostoc, e, Fermentations, r, Shigella, o, S. cerevisiae, s, Vibriosis, a, Escherichia coli, c, Vancomycin, a, Escherichia coli, r, S. cerevisiae, c, Vibriosis, c, Fermentations

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005