Culture medium

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14542 - 7 Epub 2002 Oct 22.
Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist D-serine; De Miranda J et al.; High levels of d-serine occur in the brain, challenging the notion that d-amino acids would not be present or play a role in mammals . d-serine levels in the brain are even higher than many l-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others . d-serine is synthesized by a serine racemase (SR) enzyme, which directly converts l- to d-serine . We now report that SR is a bifunctional enzyme, producing both d-serine and pyruvate in cultured cells and in vitro . Transfection of SR into HEK 293 cells elicits synthesis of d-serine and augmented release of pyruvate to culture media . We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate d-serine production by SR and elicit production of pyruvate . Experiments with recombinant enzyme reveal that Mg(2+) and ATP present in the cell extracts are physiological cofactors and increase 5- to 10-fold the rates of racemization and production of pyruvate . As much as three molecules of pyruvate are synthesized for each molecule of d-serine produced by SR . This finding constitutes a previously undescribed mechanism underlying d-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria . Our data link the production of d-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons.

Di Yi Jun Yi Da Xue Xue Bao, 2002 May, 22(5), 388 - 92
Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells; Kan WH et al.; OBJECTIVE: To understand the role of p38 mitogen-activated protein kinase (p38MAPK) in the expression of inducible nitric oxide synthase (iNOS) and NO production in human endothelial cells under the stimulation by lipopolysaccharide (LPS) . METHODS: NO level in the supernatant of the cell culture media was measured with Griess method, and iNOS protein and mRNA expressions by the cells were detected with immunofluorescence analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively . Immunoprecipitation assay was employed to examine p38 MAPK activity . RESULTS: It was shown that in comparison with the basal level of iNOS expression in cultured endothelial cells line ECV304, iNOS mRNA and protein expressions were significantly increased in the cells after LPS stimulation . In response to LPS treatment, obvious enhancement of p38 MAPK activity in ECV304 took place after the stimulation, with the peak level occurring at 15 min that maintained for approximately 45 min before gradual decline . When treated with SB203580 {4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole}, a highly specific inhibitor of p38 MAPK, significant inhibition of LPS-induced iNOS protein and mRNA expressions was observed . CONCLUSIONS: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and, inhibition of the signal transduction pathway may consequently be an effective approach to reduce the production of iNOS and other cytokines for the treatment of septic shock.

Neuropharmacology, 2002 Oct, 43(5), 877 - 88
Thiolic antioxidants protect from nitric oxide-induced toxicity in fetal midbrain cultures; Rodriguez-Martin E et al.; Nitric oxide (NO) may act as a neuroprotector or neurotoxic agent in dopamine neurons, depending on cell redox status . We have investigated the effect of several thiolic antioxidants, glutathione (GSH), its cell permeable analog GSH ethyl ester (GSHEE), and the GSH synthesis precursor L-N-acetyl cysteine (L-NAC), as well as non-thiolic antioxidants like ascorbic acid (AA) and uric acid, on NO-induced toxicity in fetal midbrain cultures . The cultures were treated for 8-24 h with neurotoxic doses of the NO donor diethylamine/nitric oxide complex sodium DEA/NO (200-400 micro M) and/or antioxidants . Thiolic antioxidants, at equimolar concentrations, added at the same time or previous to DEA/NO, protected from cell death, from tyrosine hydroxylase (TH) positive cell number decrease and from intracellular GSH depletion, induced by DEA/NO, without increasing intracellular GSH content . In these conditions, S-nitrosothiol compound formation was detected in the culture media . Protection disappeared when antioxidants were supplied 30 min after NO treatment . Nevertheless, non-thiolic antioxidants, AA and uric acid, with similar peroxynitrite scavenging activity to thiolic antioxidants, and free radical-scavenging enzymes as catalase and Cu/Zn-superoxide dismutase, which prevent extracellular peroxynitrite ion formation, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), which prevents intracellular peroxynitrite ion formation, did not rescue cell cultures from neurotoxicity induced by NO . In addition, AA exacerbated DEA/NO-induced toxicity in a dose-dependent manner from 200 micro M AA . The present results suggest that only antioxidants with thiol group exert neuroprotection from NO-induced toxicity in fetal midbrain cultures, probably by direct interaction of NO and thiol groups, resulting in NO blocking . On the other hand, some classical antioxidants, like AA, exacerbate neurotoxicity due to NO.

J Orthop Res, 2002 Sep, 20(5), 927 - 33
Expression and enzymatic activity of MMP-2 during healing process of the acute supraspinatus tendon tear in rabbits; Choi HR et al.; We investigated the spontaneous healing process of a surgically created supraspinatus tendon tear in rabbits with specific reference to the expression of matrix metalloproteinase-2 (MMP-2) and its time-course change in enzymatic activity along with the expression of tissue inhibitors of metalloproteinases (TIMPs) . A transverse, full thickness tear of the supraspinatus tendon was created and examined . Immunohistochemical analysis revealed that MMP-2 positive cells were mainly localized at both cutting ends of the tendon, and reparative tissue encroached into the gap from the bursal side . The expression of TIMP-1 was induced in the cells at not only the tendon edges but also the reparative tissue during the healing process . TIMP-2 was constitutively expressed in both the tendon and the reparative tissue . Gelatin zymography using tissue culture media demonstrated latent and active forms of MMP-2 and characteristic time-linked changes of the enzymatic activity . Western blotting confirmed the bands as the latent form of MMP-2 . These results suggest that MMP-2 is expressed and activated during the healing process of acute supraspinatus tendon tear and can play an important role in the remodeling process.

Zhonghua Zheng Xing Wai Ke Za Zhi, 2002 Jul, 18(4), 234 - 6
{Substance P up-regulates the TGF-beta 1 mRNA expression of human dermal fibroblasts in vitro}; Hu D et al.; OBJECTIVE: To investigate the role of substance P in the formation of hypertrophic scar . METHODS: Dermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt . The effect of substance P on proliferation of fibroblasts was measured by MTT assay . Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis . RESULTS: Substance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media . By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P . The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt . CONCLUSION: The results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring.

Anal Biochem, 2002 Oct 1, 309(1), 79 - 84
How to make tetracycline-regulated transgene expression go on and off; Rennel E et al.; Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals . While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline . Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines . Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media . The released doxycycline reached sufficiently high levels to completely suppress transgene expression . The effect was not dependent on cell type or the nature of the transgene . However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline . The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results . These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.

J Matern Fetal Neonatal Med, 2002 Jan, 11(1), 11 - 7
Thrombin-enhanced matrix metalloproteinase-1 expression: a mechanism linking placental abruption with premature rupture of the membranes; Rosen T et al.; OBJECTIVE: Given the strong clinical association between the decidual hemorrhage of placental abruption and subsequent preterm premature rupture of the membranes, we assessed the effects of thrombin on the expression of the potent interstitial collagenase, matrix metalloproteinase-1 (MMP-1), in cultured endometrial stromal and decidual cells . STUDY DESIGN: Stromal cells derived from predecidualized cycling endometrium and decidual cells from term decidua were cultured in a defined medium containing estradiol, to mimic the hormonal milieu of the non-pregnant proliferative phase, or estradiol plus medroxyprogesterone acetate (MPA), to mimic the hormonal milieu of pregnancy, in the presence and absence of thrombin . Culture media were examined for MMP-1 protein levels and cell lysates were examined for steady-state MMP-1 mRNA levels . RESULTS: MPA strongly inhibited MMP-1 levels in endometrial stromal and term decidual cells . However, thrombin overcame this suppression, producing MMP-1 levels that were several-fold higher than control levels . CONCLUSION: Extrapolation of thrombin-enhanced MMP-1 expression in cultured endometrial stromal and decidual cells to the in vivo pregnant state provides an explanation for the strong association between placental abruption and preterm membrane rupture.

Biochem Biophys Res Commun, 2002 Oct 18, 298(1), 10 - 16
A double-strand decoy DNA oligomer for NF-kappaB inhibits TNFalpha-induced ICAM-1 expression in sinusoidal endothelial cells; Shibuya T et al.; Altered gene expression of liver sinusoidal endothelial cells (SECs) is associated with impaired immune response . Here we report that the decoy technique effectively suppresses TNFalpha-induced ICAM-1 expression in SEC . An NF-kappaB decoy (NF-kappaB31: 5(')-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3), which contains a consensus sequence for NF-kappaB, was complexed to PLL-g-HA {hyaluronate-grafted poly(L-lysine) copolymer} that permits transfer of exogenous DNA selectively to the SEC . The PLL-g-HA/NF-kappaB31 complex was added to the culture media of LSE cells, a human SEC-derived cell line . Then, cells were stimulated with TNFalpha (5ng/mL) . PLL-g-HA/NF-kappaB31, but not control oligodeoxynucleotides having a reverse or scrambled sequence, inhibited the intranuclear localization of NF-kappaB induced by TNFalpha, with almost complete inhibition at 2.5microg/mL as DNA . NF-kappaB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells . The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Jul, 22(7), 624 - 5
In vitro culture and study of the biological characteristics of rabbit keratocytes; Huo XK et al.; OBJECTIVE: To improve the technique for culturing rabbit keratocytes in vitro and investigate the biological characteristics of these cells . METHODS: Fresh rabbit corneas were obtained and the epithelial and endothelial cells were removed after digestion with trypsin . The stroma was rinsed, minced, and incubated in DMEM/F12 medium supplemented with 10% fetal bovine serum and the biological characteristics of the keratocytes were observed with MTT assay and compared with those of the cells in serum-free culture media . RESULT: On about the third day of incubation, some keratocytes germinated from the stromal tissues and migrated onto the flask surface presenting fibroblast-like arrangement with spindle-shaped appearance . The keratocytes became confluent after 10 day's incubation and the peak of cell proliferation occurred on day 8 in the presence of serum in the media, while in the absence of serum, the peak took place on day 10 . CONCLUSION: The method improved for in vitro keratocyte culture is convenient and effective, and the presence of serum in the media may to some degree affect the growth of the keratocytes.

Clin Chest Med, 2002 Sep, 23(3), 623 - 32, vii
Pulmonary disease caused by rapidly growing mycobacteria; Daley CL et al.; The rapidly growing mycobacteria (RGM) differ from slow-growing mycobacteria such as Mycobacterium tuberculosis by virtue of their more rapid growth in culture media and their in vitro resistance to standard antituberculosis drugs . The RGM can produce numerous infections including chronic lung disease . The most common causes of pulmonary disease are Mycobacterium abscessus and Mycobacterium fortuitum . This article reviews the management of patients with lung disease caused by RGM.

Antibiot Khimioter, 2002, 47(4), 3 - 6
{Concentration on Diapak C 16 capsules of lovastatin, mevinolinic acid and other inhibitors of biosynthesis of sterins produced by Penicillium citrinum 89}; Baranova NA et al.; The method of lovastatine and mevinolinic acid known as competitive inhibitors of HMG-CoA-reductase and produced by micromycetes was elaborated . The inhibitors from diluted water solutions were fully absorbed on Diapak C16 patrons . The rate of inhibitors elution from the patrones was more than 95 per cent . Patrons may be used for concentration of lovastatine group inhibitors from the culture media . Inhibitors synthesis by the Penicillium citrinum 89 was investigated in dynamics with the use of Diapak C16 patrones . It was shown that UV-spectrum of inhibitor produced by P . citrinum 89 was identical with compactin spectrum and had absorbance maximum at 230, 237 and 247 nm.

Exp Dermatol, 2002 Oct, 11(5), 413 - 20
Organotypic keratinocyte coculture using normal human serum: an immunomorphological study at light and electron microscopic levels; Hinterhuber G et al.; Organotypic human skin equivalents of keratinocytes and fibroblasts embedded in collagen matrix have been the subject of studies dealing with various culture conditions . Development of standardized living skin equivalents using defined culture media containing respective supplements can provide important instruments of investigation in skin biology . In addition, tissue engineering has created human skin substitutes for treatment of acute and chronic wounds . In our study, we generate a modified organotypic human skin equivalent using normal human serum instead of fetal calf serum (FCS) . This living skin equivalent shows regular stratification of the epidermis and the dermal-epidermal junction zone at the light and electron microscopic level after 1 and 3 weeks of coculture . Indirect immunofluorescence reveals regular expression of differentiation antigens and the major structural proteins collagen IV, laminin 5 and the integrin chains alpha 6 and beta 4 at the dermo-epidermal junction zone . Immunoelectron microscopy demonstrates expression of collagen IV, alpha 6 and beta 4 integrin after 1 and 3 weeks of coculture . This organotypic skin model could be the basis for autologous skin grafting for acute or chronic wounds using autologous serum as well as patients' keratinocytes and fibroblasts, thus minimizing the risk of transmitting infectious agents.

J Clin Endocrinol Metab, 2002 Oct, 87(10), 4775 - 81
Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells; Movassat J et al.; In addition to stimulating insulin secretion, glucagon-like peptide and its long-acting analog exendin 4 have been reported to increase beta-cell mass by both differentiation/neogenesis of precursor cells and enhanced replication of existing beta-cells . Here, we investigated the effect of exendin 4 in the growth and differentiation of beta-cells from undifferentiated precursors in islet-like cell clusters (ICCs) derived from human fetal pancreases . Our results show that the addition of exendin 4 to the culture media stimulates PDX 1 expression in ICCs as shown by immunofluorescence staining . The up-regulation of PDX 1 was not accompanied by changes in insulin expression because we did not find a significant difference in the number of insulin-positive cells in the exendin 4-treated ICCs, compared with controls . We also tested the effects of exendin 4 in the glucose-induced insulin secretion of human ICCs transplanted under the kidney capsule of athymic rats . In the exendin 4-treated rats (given ip during 10 d) 8 wk after the beginning of the treatment, insulin was released in response to glucose as detected by the measurement of circulating human C-peptide . In control (saline-treated) rats, the basal levels of human C-peptide did not change significantly after glucose stimulation . Thus, exendin 4 induces functional maturation of fetal beta-cells in response to glucose . In these rats, serial sections of the kidney-bearing grafts were examined histologically for insulin containing cells . We found a significant increase in beta-cell number, compared with the control rats . Overall, these results show that in vivo exendin 4 causes growth and differentiation of human fetal beta-cells from undifferentiated precursor cells . It also accelerates the functional maturation of fetal beta-cells as evidenced by their glucose-stimulated insulin secretion.

Thromb Haemost, 2002 Oct, 88(4), 632 - 8
Protein C deficiency caused by homozygosity for a novel PROC D180G mutation--in vitro expression and structural analysis of the mutation; Lind B et al.; Homozygosity for a novel D180G mutation in the protease domain of protein C, associated with plasma protein C activity and antigen levels of 8% of normal was identified in a thrombosis prone family . Transient expression of protein C in HK-293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein as compared with wild-type protein was reduced by 79% while the intracellular contents were similar . Computer analysis of the X-ray structure of activated protein C and of a theoretical model of the zymogen predicts that the mutation destabilises the molecule locally . Our results are compatible with a relatively unstable mutant molecule that could be trapped inside the cell and degraded . However, if secreted the mutant molecule could have a relatively normal catalytic activity and structure consistent with the plasma levels of protein C activity and the late onset of thrombosis.

Lett Appl Microbiol, 2002, 35(4), 272 - 5
The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link; Santos IM et al.; AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum . METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying . Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation . Citrinin was detected in all cultures for all preservation techniques on YES . The patulin profiles obtained differed with strain and culture media used . CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains . There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ . SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used.

Mol Biotechnol, 2002 Sep, 22(1), 87 - 98
Downstream processing in the biotechnology industry; Kalyanpur M; The biotechnology industry today employs recombinant bacteria, mammalian cells, and transgenic animals for the production of high-value therapeutic proteins . This article reviews the techniques employed in this industry for the recovery of these products . The methods reviewed extend from the centrifugation and membrane filtration for the initial clarification of crude culture media to the final purification of the products by a variety of membrane-based and chromatographic methods . The subject of process validation including validation of the removal of bacterial and viral contaminants from the final products is also discussed with special reference to the latest regulatory guidelines.

Res Reprod . 1977 Sep;9(5):4.
Albumin and the acrosome change in mammalian spermatozoa; Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber; Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United KingdomWe have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development . In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes . Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media . The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate . Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL . Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase . However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL . It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.

J Biol Chem, 2002 Dec 20, 277(51), 49831 - 40 Epub 2002 Sep 18.
Myostatin inhibits myoblast differentiation by down-regulating MyoD expression; Langley B et al.; Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts . In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3 . In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media . Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation . The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation . Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression . Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin . Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes . Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

Microsc Res Tech, 2002 Sep 15, 58(6), 488 - 95
Melanization stimulating factor (MSF) and melanization inhibiting factor (MIF) in the integument of fish; Zuasti A; The present studies were directed to demonstrate that adult fish skin contains putative factors that affect chromatophore and/or chromatoblast function . This hypothesis is based upon the possibility that hypo and/or hyperpigmented areas of the skin are so pigmented because of the localized expression of intrinsic factors that are either stimulatory or inhibitory to the differentiation of specific pigment cell types . In all the morphological and biochemical experiments carried out, we used culture media conditioned by dorsal (DCM) or ventral (VCM) skin from different species of fish . Both DCM and VCM were capable of stimulating differentiation of melanophores in neural crest explants . While the stimulation of melanization is an activity present in both dorsal and ventral skin, an inhibitory activity is also present in ventral skin at such a concentration that it overrides the stimulatory activity afforded by DCM . With biochemical assays, we demonstrated that the three important sequential enzymatic steps in melanogenesis are all stimulated by the conditioned media in a dose-dependent manner and this results in an increase in the amount of melanin present in cultured cells . The results of our investigations provide strong evidence that there are intrinsic pigment cell regulatory factors in the integument of fish, the inhibitory activity being stronger in the ventrum, and that those factors strongly influence, perhaps even determine, the pigment patterns of fish .

Theriogenology, 2002 Oct 1, 58(6), 1081 - 95
The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes; Kawahara M et al.; A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer . We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation . In Experiment 1, we examined activation rates of in vitro matured oocytes . These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment . In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation) . In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs) . In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, {+} 0.0%, {-} 60.7% at 24 h after treatment, P < 0.05) . In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes . At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions . At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time . In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, {+} 28.5%, {-} 66.4% at 24 h after treatment, P < 0.05) . The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation.

Skin Pharmacol Appl Skin Physiol, 2002 Sep-Oct, 15(5), 335 - 41
Trioxsalen in the presence of UVA is able to induce nuclear factor kappa B binding activity in HaCaT keratinocytes; Sanchez Ruderisch H et al.; It has been described that treatment of cells with high dose psoralen and UVA induce the production of reactive oxygen species (ROS) leading to DNA damage . Transcription factor nuclear factor kappa B (NFkappaB) plays a crucial role in regulating not only cell growth but also cell differentiation, and ROS seem to be partly involved in these mechanisms . The aim of this research was to find out the effect of a combined treatment with trioxsalen (TMP)/UVA on NFkappaB binding activity in HaCaT keratinocytes . HaCaT keratinocytes were treated with 27 microg/l TMP . This concentration did not affect the proliferation rates, nor was it toxic, as shown by cytotoxicity assays . After treatment with TMP with or without UVA (1 J/cm(2)), NFkappaB binding activity in nuclear protein extracts was measured by electrophoretic mobility shift assays . The effect on cytokines and cytokine receptor genes was investigated using cDNA expression arrays . An inhibitory effect on NFkappaB binding activity was found between 30 and 60 min after TMP supplementation of the culture media . UVA irradiation induced a 2-fold increase in NFkappaB binding activity in TMP supplemented HaCaT keratinocytes compared with the non-irradiated control . In addition, NFkappaB binding activity was higher after UVA irradiation with TMP than in UVA irradiated cells in the absence of TMP . TGF-alpha, IL-1R, IL-2Ralpha, IL-12beta and PDGF expression was induced by UVA . However, all of them except PDGF were inhibited by combined TMP/UVA treatment . Using an inhibitor of NFkappaB activation, we found out that under these conditions, these cytokines or cytokine receptor genes are apparently not regulated by NFkappaB . Our results indicate that a combined TMP/UVA treatment of HaCaT keratinocytes induces NFkappaB binding activity, and that this is a synergistic effect . The investigated cytokines, and cytokine receptor genes do not seem to be NFkappaB regulated; however, TMP shows anti-inflammatory capacities in vitro .

J Neurosci Res, 2002 Oct 1, 70(1), 36 - 45
Inhibition of insulin-like growth factor I activity contributes to the premature apoptosis of cerebellar granule neuron in weaver mutant mice: in vitro analysis; Zhong J et al.; Evidence from transgenic mice and cultured cerebellar neurons supports an important role for insulin-like growth factor I (IGF-I) in the formation of cerebellar cytoarchitecture . To understand IGF-I's function during cerebellar development, we examined the involvement of IGF-I in the premature apoptosis of granule neurons derived from the cerebella of weaver (wv) mutant mice . Before their demise, wv granule neurons increased the expression and secretion of IGFBP5 in a gene dose-dependent manner . Because IGFBP5 may interfere with the interaction of IGF-I and its receptor, the abnormally high IGFBP5 levels in wv granule neurons suggest that a lack of IGF-I activation may contribute to their premature apoptosis . This hypothesis is supported by a gene dose-dependent decrease in IGF-I receptor (IGF-IR) phosphorylation . More importantly, there is a parallel gene dose-dependent decrease in Akt activity, which was inversely correlated with the activity levels of caspase 3 . On the other hand, adding IGFBP5 antibody into culture media increased the survival of wv granule neurons, whereas adding IGFBP5 decreased the survival of wild-type granule neurons . To delineate the interaction between IGF-I and IGFBP5 on wv granule neurons, we examined neuronal survival after treating with IGF-I, des(1-3) IGF-I, or IGFBP5 antibody . At the same concentration, des(1-3) IGF-I was more effective than IGF-I in promoting survival, in increasing Akt activity, and in decreasing caspase 3 activity . These results indicate that IGF-I's actions on wv granule neurons are normally inhibited by excess IGFBP5, and sufficient IGF-I receptor activation rescues wv granule neurons via stimulating the Akt signaling pathway .

Inflamm Res, 2002 Aug, 51(8), 427 - 33
Differential effects of nonsteroidal antiinflammatory drugs on the IL-1 altered expression of plasminogen activators and plasminogen activator inhibitor-1 by articular chondrocytes; Sadowski T et al.; OBJECTIVE: To determine the in vitro effects of several nonsteroidal antiinflammatory drugs on the IL-1 altered expression and activity of tPA, uPA and PAI-1 by articular chondrocytes . METHODS: Bovine chondrocytes were cultured in alginate gel beads . Cells were treated with IL-1alpha in the presence or absence of drugs at various concentrations . Expression of mRNA for the plasminogen activators (uPA and tPA) and their inhibitor (PAI-1) were analyzed by RT-PCR-ELISA . The protein content of PAI-1 in culture media was deter mined by ELISA . PA activity was measured by a functional assay . RESULTS: All tested NSAIDs dose dependently inhibited the IL-1 induced mRNA expression of tPA, whereas only indomethacin and tiaprofenic acid were also able to reduce the expression of uPA . Expression of PAI-1 was elevated by IL-1 without an accompanying increase in secreted amounts of the inhibitor . Indomethacin, naproxen and tiaprofenic acid stimulated the release of PAI-1 into culture media, whereas meloxicam also induced expression of PAI-1 above IL-1 stimulated levels . CONCLUSION: In conclusion, our studies indicate that NSAIDs preferentially inhibit tPA expression by bovine articular chondrocytes . By increasing the production of PAI-1 at therapeutical concentrations meloxicam could reduce PA activity, whereas the other NSAIDs tested mainly enhanced the release of this inhibitor from the extracellular matrix . In how far this would affect the enzyme-inhibitor balance within cartilage has to be determined in further studies.

Vet Rec, 2002 Aug 31, 151(9), 260 - 5
Effect of insulin, transferrin and selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in serum-free, semidefined media; Raghu HM et al.; The in vitro development of buffalo oocytes up to the blastocyst stage was studied in serum-free, semidefined media containing bovine serum albumin, follicle-stimulating hormone (FSH), insulin, transferrin and selenium (ITS) and epidermal growth factor (EGF) . In experiment 1, oocytes aspirated from abattoir-derived ovaries were cultured in eight serum-free, semidefined culture media containing different combinations of these four factors . In experiment 2, the maturation of buffalo oocytes and the development of the embryos were compared in a complex co-culture system and in the serum-free, semidefined media . Supplementation with FSH and EGF significantly (P < 0.05) increased the maturation rates of buffalo oocytes, and the yield of blastocysts was higher (P < 0.05) in media containing EGF and ITS . The yield of blastocysts was lower in the serum-free semidefined media (P < 0.05) than in the complex co-culture system.

Neurotoxicology, 2002 Jul, 23(2), 147 - 57
Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation; Roth JA et al.; Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations . Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death . To accomplish this, we have utilized rat pheochromocytoma (PC12) cells as a model since they possess much of the biochemical machinery associated with dopaminergic neurons . Mn, like nerve growth factor (NGF), can induce neuronal differentiation of PC12 cells but Mn-induced cell differentiation is dependent on its interaction with the cell surface integrin receptors and basement membrane proteins, vitronectin or fibronectin . Similar to NGF, Mn-induced neurite outgrowth is dependent on the phosphorylation and activation of the MAP kinases, ERK1 and 2 (p44/42) . Unlike NGF, Mn is also cytotoxic having an IC50 value of approximately 600 microM . Although many apoptotic signals are turned on by Mn, cell death is caused ultimately by disruption of mitochondrial function leading to loss of ATP . RT-PCR and immunoblotting studies suggest that some uptake of Mn into PC12 cells depends on the divalent metal transporter 1 (DMT1) . DMT1 exists in two isoforms resulting from alternate splicing of a single gene product with one of the two mRNA species containing an iron response element (IRE) motif downstream from the stop codon . The presence of the IRE provides a binding site for the iron response proteins (IRP1 and 2); binding of either of these proteins could stabilize DMT1 mRNA and would increase expression of the +IRE form of the transporter . Iron and Mn compete for transport into PC12 cells via DMT1, so removal of iron from the culture media enhances Mn toxicity . The two isoforms of DMT1 (+/-IRE) are distributed in different subcellular compartments with the -IRE species selectively present in the nucleus of neuronal and neuronal-like cells.

Ultrason Sonochem, 2002 Sep, 9(4), 197 - 203
Effects of dissolved gases and an echo contrast agent on ultrasound mediated in vitro gene transfection; Ogawa R et al.; The effects of acoustic cavitation on in vitro transfection by ultrasound were investigated . HeLa cells were exposed to 1.0 MHz continuous ultrasound in culture media containing the luciferase gene . Transfection efficiency was elevated when an echo contrast agent, Levovist was added or air was dissolved in the medium . When cells were sonicated in medium saturated with Ar, N2 or N2O which have different gamma values (Cp/Cv), or were saturated with He, Ar or Ne with different thermal conductivities, the effectiveness for the dissolved gases in the ultrasound mediated transfection was Ar > N2 > N2O or Ar > Ne > He, respectively . When free radical formation in water by ultrasound was monitored as a measure of inertial cavitation, it was similarly affected by dissolved gases . These results indicate that the efficiency of ultrasound mediated transfection was significantly affected either by occurrence of or by modification of inertial cavitation due to various gases.

Anal Bioanal Chem, 2002 Sep, 374(1), 64 - 8 Epub 2002 Jul 23.
Direct analysis of nine pharmaceuticals in culture media by use of cartridge separation with electrospray mass spectrometric detection; Li XF et al.; A 2-cm cartridge has been used for separation before electrospray mass spectrometric analysis of pharmaceutical compounds in cell culture media, alleviating the need for sample extraction and desalting procedures . Nine representative pharmaceuticals listed in the biopharmaceutical classification system (BCS) were chosen as the candidate compounds and Hank's balanced salt solution with Hepes buffer (HBSS-Hepes buffer) was used as the cell-culture medium in an effort to study permeability of chemicals through cell monolayers . Effects of several conditions, e.g . pH and buffer concentration in the mobile phase, flow rate, and temperature on separation efficiency were examined . The nine pharmaceuticals were separated within 2 min by use of a 2-cm C(8) cartridge . Relative standard deviations (RSD) from repeated analysis within the same day or over five days were 0.03-0.2% for retention times and 0.6-5.3% for peak areas; antipyrine was used as internal standard . Calibration curves based on peak-area measurements were linear over the range 0.1-20 micro mol L(-1) . The HBSS-Hepes buffer did not interfere with separation and detection; identical separation and peak intensity were obtained when the samples were separately prepared in distilled water or in the culture medium.

Domest Anim Endocrinol, 2002 Oct, 23(3), 383 - 96
Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe; McGuire WJ et al.; Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNtau) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized . Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17beta (E(2)) and/or progesterone (P(4)) for 14 days, in day 14 cyclic and day 14 pregnant ewes . Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E(2)- and E(2)/P(4)-treated ewes than that of control or P(4)-treated ovariectomized ewes . Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E(2)- and E(2)/P(4)-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher . GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E(2)/P(4)-treated ovariectomized ewes were 3-fold higher than those of control, E(2)- and P(4)-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher . Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E(2)/P(4)-treated ovariectomized, cyclic and pregnant ewes . Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques . Data indicate that combined E(2) and P(4) treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes . These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF.

Environ Toxicol Chem, 2002 Sep, 21(9), 1788 - 95
Use of anodic stripping voltammetry in predicting toxicity of copper in river water; Wang Z et al.; The labile concentration and toxicity of Cu as influenced by alkalinity and different concentrations of ethylenediaminetetraacetic acid (EDTA) and naturally derived fulvic acid (FA) were determined by bioassays carried out in the culture media for Daphnia magna (D . magna) . The labile concentration of Cu was obtained by differential pulse anodic stripping voltammetry with a double-acidification method (DAM-DPASV) . Changes in water alkalinity did not affect the labile concentration of Cu, but increase in alkalinity did reduce the mortality of D . magna . In the presence of EDTA and FA, both labile concentration of Cu and mortality were reduced . By excluding Cu-carbonate complexes from the labile concentration, a bioavailable concentration of Cu ({Cu*}) was obtained and was used to predict the acute toxicity of Cu on D . magna . For natural waters, the labile concentration of Cu was measured by DAM-DPASV, and {Cu*} was calculated using MINTEQ A2 software (developed by the U.S . Environmental Protection Agency) based on the anion composition of waters . This procedure was tested for waters and sediment elutriates sampled from the Le An River (Jiangxi Province, China) that were severely polluted by the discharges from a copper mine . The results showed that {Cu*} was a good indicator for Cu toxicity and could be used under field conditions.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(3), 303 - 309
Expression of a Ligninase by a New Hybrid Baculovirus with Expanded Host Range; Lin QY et al.; We have generated and purified a new recombinant baculovirus with expanded host range . AcNPV DNA and BamHI-digested BmNPV DNA were co-transfected into Spodoptera frugiperda SF21 cells . Progeny viruses were used to infect BmN cells, which are normally resistant to AcNPV infection, in order to screen for recombinant viruses with cross-infectivity . A recombinant virus was isolated by plaque purification and analyzed . This isolate was able to infect, replicate and produce polyhedrin in both the SF21 and BmN cells . DNA restriction endonuclease analysis showed that it was a hybrid (HyNPV) of AcNPV and BmNPV . Co-transfection of this HyNPV virus with a transfer vector containing a ligninase H8 gene insert into SF21 cells yielded a recombinant baculovirus expressing the ligninase . When this recombinant baculovirus was used to infect either SF21 or BmN cells, ligninase proteins could be found in the lysed cells as well as in the cell culture media . We have thus demonstrated that this hybrid virus can be utilized in the generation of recombinant baculovirus expressing foreign proteins in two host cells which normally can not be infected by AcNPV nor BmNPV.

Anesthesiology, 2002 Sep, 97(3), 630 - 5
Growth cone collapsing effect of lidocaine on DRG neurons is partially reversed by several neurotrophic factors; Radwan IA et al.; BACKGROUND: Local anesthetics were suggested to have a potential for neurotoxicity in both clinical reports and laboratory experiments . Growing neurons have been shown to be susceptible to the toxic effects of local anesthetics in culture . These findings have generated the interest in factors that would rescue the neurons affected by the neurotoxicity of local anesthetics . METHODS: Primary cultured dorsal root ganglia were isolated from age-matched chick embryos and exposed to lidocaine . After 60 min of exposure, the culture media were replaced to wash out the lidocaine . Neurotrophic factors (NTFs)-brain-derived neurotrophic factor, glial-derived neurotrophic factor, or neurotrophin 3-were added to the replacement media to examine the capacity of these NTFs to support the reversibility of the lidocaine-induced growth cone collapse . The growth cone collapse assay was applied a quantitative method of assessment . RESULTS: When any of the three NTFs was added to the replacement media at a minimum concentration of 10 ng/ml, significantly high reversibility of the lidocaine-induced growth cone collapse was observed, especially at 48 h after washout (P < 0.05) . At that time point, there was no significant difference between the values of growth cone collapse percentage in the cells that were exposed to lidocaine and supported by the NTFs after the washout, and the control cells (not exposed to lidocaine) (P > 0.05) . CONCLUSION: The NTFs-brain-derived neurotrophic factor, glial-derived neurotrophic factor, and neurotrophin 3-were demonstrated to support the reversibility of lidocaine-induced growth cone collapse in primary cultured sensory neurons, an effect that was concentration- and time-dependent . Because similar effects were observed after tetracaine washout, the supporting effects of NTFs may not be specific to lidocaine.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Sep, 30(3), 340 - 2
{The influential factors in using cadmium reduction method for measurement of the stable products of NO--nitrates and nitrites}; Gu L et al.; To establish an efficient assay method for detecting nitric oxide indirectly, we compared the effects of cadmium filing, 5 mmol/L and 80 mmol/L copperized cadmium filing on the measurement of nitrates and nitrites (the stable products of NO) using the cadmium reduction method . The results demonstrated that cadmium filing was the most efficient cadmium preparations (P < 0.001) . It's mean reduction rate was 96%, and it showed stronger reduction effectiveness in deproteinization cell culture media . The results indicated that cadmium filing has strong anti-interference capacity in biological fluid . We recommend the cadmium filing reduction method because it is simple, practical, inexpensive, highly efficient and can be performed in ordinary laboratories.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Sep, 30(3), 233 - 5
{Effects of insulin on synthesis and secretion of apolipoproteins A I, A II, B100, C III and E by cultured HepG2 cells}; Wang H et al.; This study was intended to observe the effects of insulin on synthesis and secretion of apolipoproteins by hepatic cells . The HepG2 cells were cultured in RPMI 1640 media with bovine insulin (1 microgram/ml media and 10 micrograms/ml media) for 72 hours, and the apolipoproteins contents in cultured media were measured by radioimmunodiffusion assay (RID) kits developed by authors' research laboratory . 40 fold lyophilizely condensed culture media were used for the assays . The results showed that the relative amounts of apo A I, A II, B100, C III and E secreted by HepG2 cells detected by this method were 824 +/- 27, 813 +/- 24, 4875 +/- 82, 597 +/- 74 and 97 +/- 4 ng/mg cell protein, respectively . When 1.0 microgram/ml medium of bovine insulin was added into the culture media, apo A I and apo E secretion remained unchanged . When compared with the control, while apo A II tended to decrease, apo C III tended to increase, and apo B100 secretion decreased by 21.2%; when 10.0 micrograms/ml medium of insulin was added, apo A I, A II and B100 secretion decreased by 6.9% (P < 0.05), 44.3% (P < 0.01) and 29.3% (P < 0.01) respectively, while apo C III secretion increased by 51.8% (P < 0.01) . These results support certain parts of the hypothesis of pathogenesis of Chinese endogenous hypertriglyceridemia proposed by Bing-Wen Liu.

Poult Sci, 2002 Aug, 81(8), 1191 - 8
The effect of hepatocyte growth factor on turkey satellite cell proliferation and differentiation; Zeng C et al.; The effect of hepatocyte growth factor (HGF) on turkey satellite cell proliferation and differentiation was examined in cell culture . Satellite cell clones were established from one muscle of an individual turkey . The results showed that HGF is a potent activator and mitogen of turkey satellite cells and embryonic myoblasts with maximal stimulation at 1 ng/mL . HGF is also an inhibitor of differentiation of turkey satellite cells . Heterogeneity in the responsiveness to HGF in the turkey satellite cell population was observed between clones selected for fast (Early) or slow (Late) rates of proliferation . However, two other Early clones exhibited responses similar to those of two other Late clones . When combined with insulin-like growth factor (IGF) and fibroblast growth factor (FGF), singularly or in combination, HGF did not exert any additive or synergistic effects on Early or Late clone proliferation . Whereas when combined with IGF, FGF, and platelet-derived growth factor (PDGF), HGF significantly stimulated proliferation of the Late clone but not the Early clone . Addition of anti-HGF antibody to culture media diminished proliferation and provided evidence of autocrine production of HGF by turkey satellite cell cultures . Heterogeneity also exists in the turkey satellite cell population with respect to autocrine production of HGF.

Bioessays, 2002 Sep, 24(9), 845 - 9
Quiet please, do not disturb: a hypothesis of embryo metabolism and viability; Leese HJ; This review uses nutritional markers of normal and impaired development to address the question; what makes a viable mammalian preimplantation embryo? Resolution of this question is important to ensure the long-term safety of embryo-based biotechnologies in man and domestic animals, the optimisation of embryo production and culture conditions and the development of methods to select viable embryos for replacement . After considering the nutrition of embryos and somatic cells, and the phenomenon of caloric restriction, it is concluded that preimplantation embryo survival is best served by a relatively low level of metabolism; a situation achieved by reducing the concentrations of nutrients in culture media and encouraging the use endogenous resources .

Biotechnol Bioeng, 2002 Oct 5, 80(1), 73 - 83
Determination of oxygen gradients in engineered tissue using a fluorescent sensor; Kellner K et al.; Nutrient and oxygen supply of cells are crucial to tissue engineering in general . If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely . Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution . The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues . Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period . Cylindrical tissue samples were immobilized on top of the sensors . By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined . We applied this technique to cartilage explants and to tissue-engineered cartilage . For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples . Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day . Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function . Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions . Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center . The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures .

Parasitol Res, 2002 Oct, 88(10), 946 - 9 Epub 2002 Jun 14.
Acid phosphatase activity in excretion/secretion products from Heligmosomoides polygyrus adults: an indicator of the physiological status of the worms; Martinez-Grueiro MM; Acid phosphatase (AP) activity was detected in 24 h culture media from adult Heligmosomoides polygyrus . Female and male excretion/secretion products showed similar specific activity . For both, the AP had a pH optimum of 4.0 and was inhibited by sodium fluoride, tartaric acid, and sodium orthovanadate . The release of AP by adult worms was significantly inhibited by adverse incubation conditions (temperatures of 20 degrees C and 4 degrees C), known physiological perturbers ( t-butylhydroperoxide and sodium azide), and broad spectrum anthelmintics (albendazole, levamisole, morantel, and ivermectin) . These results indicate that the AP activity level in the culture medium may be an indicator of the physiological status of the worms.

J Neurophysiol, 2002 Sep, 88(3), 1475 - 90
Differential involvement of Ca(2+) channels in survival and neurite outgrowth of cultured embryonic cockroach brain neurons; Benquet P et al.; The contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents . VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil . The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about -30 and -20 mV in high-potassium culture media) . As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM . The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network . In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I(Ba) . This effect could not be prevented by addition of fetal calf serum to the culture medium . A similar effect was obtained using intra- or extracellular calcium chelating agents (10 microM BAPTA-AM or 10 mM EGTA) . Quite unexpectedly, blockade of the P/Q-like (omega-Aga WA-sensitive) component of the calcium current by 500 nM of omega-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism . As expected from their lack of effect on I(Ba), isradipine, nifedipine, and omega-CgTx GVIA did not induce cell death . When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion . In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM . However, these agents, as well as omega-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons . This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites . These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons . The differential contribution of the P/Q-like and R-like (omega-Aga WA-sensitive) calcium channels in these processes is discussed.

J Biochem (Tokyo), 2002 Sep, 132(3), 493 - 9
Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E; Liu J et al.; Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif . Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis . Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells . The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions . However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively . The K(m) values of these two mutants were similar to those of the wild-type enzyme, but their k(cat) values were greatly decreased . The K(i) values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same . The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values . These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product.

J Clin Microbiol, 2002 Sep, 40(9), 3499 - 501
Early detection of negative BACTEC MGIT 960 cultures by PCR-reverse cross-blot hybridization assay; Romano L et al.; We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures . Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay . PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system . Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay . In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures.

Life Sci Space Res, 1965, 3, 139 - 41
Bacterial ecologies in limonite; Vishniac W; Limonite (Fe2O3 . nH2O) may be a constituent of the Martian surface . We have prepared culture media with ferric hydroxide as an electron acceptor . One medium contained ethanol, another gaseous hydrogen and carbon dioxide . Bacterial growth without light and oxygen suggests that ferric iron serves as a terminal respiratory electron acceptor . The oxidation of ferrous hydroxide may be carried out by photosynthetic bacteria . A ferrous-ferric couple may thus support bacterial respiration and photosynthesis in the absence of oxygen . This cycle may account for the dark markings of Mars.

Med Lav, 2002 May-Jun, 93(3), 267 - 78
{In vitro study of the nephrotoxic mechanism of mercuric chloride}; Aleo MF et al.; OBJECTIVES: Mercury (Hg), one of the most diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of which with unique characteristics of target organ specificity . Exposure to Hg vapour and to organic mercurials specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds . Despite the increasing number of studies, the molecular bases of the nephrotoxic potential of Hg has not, up to now, been clarified, even if there is evidence suggesting that the ability of the metal to interact with proteins (thiol groups) or to generate oxygen radicals may play a major role . Within this context, the aim of the present study was to investigate, in vitro, the mechanism(s) of the early nephrotoxic potential of mercury chloride (HgCl2), one of the most diffused and biologically active mercury (Hg2+) compounds . For this purpose, two kidney-derived in vitro systems (the MDCK and the LLC-PK1 cell lines) were tested for their sensitivity to the salt, and MDCK was chosen as the most suitable in vitro model for our study . As possible biological markers of the organ-specific toxicity of the metal we analysed: i) critical biochemical parameters related to oxidative stress conditions (effect of Hg2+ on the anti-oxidant status of the cell), and ii) gap-junctional function (GJIC) . METHODS: Classical toxicity tests (MTT and NR) were used for assessing the sensitivity (IC50) of LLP-CK1 and MDCK cell lines to the mercuric salt . Complete solubilisation of the salt in the culture media was verified by inductively coupled plasma mass spectrometry (ICP-MS) . The influence of the metal on cell growth rate and viability were evaluated by conventional proliferation assays . For the following mechanistic studies, cells were exposed for different time periods (4 to 72 hours) to non-cytotoxic (0.1-50 microM) HgCl2 concentrations . The biochemical analysis of the pro-oxidant properties of the mercuric compound was performed by the measurement of anti-oxidant cellular defences against H2O2 {catalase (Cat), glutathione peroxidase (Gpx), and total glutathione (GSH)} . The influence of the metal on the GJIC capacity of MDCK cells was assessed by the "microinjection/dye-coupling" assay . RESULTS: Among the two kidney-derived in vitro systems, MDCK cell line was the most specifically sensitive to the toxic effect of HgCl2: it was, consequently, chosen as a "tubular cell model" for the following experimental steps . Tested for various time periods at increasing concentrations, the HgCl2 effect on MDCK cell proliferation and viability was found to be time- and dose-related . For concentrations < or = 50 microM, HgCl2 inhibits MDCK cell growth rate, being this effect significant (> 50% in respect to untreated controls) from the 24th from the beginning of the treatment, while, for concentrations > 50 microM, the metal causes cell death . Concerning the influence of HgCl2 on MDCK anti-oxidant defences, the most interesting results were obtained by analysing the influence of the mercury salt on the GSH cell content and Gpx activity . Both were, in fact, significantly affected by the presence of the mercury ion . HgCl2 also induced a rapid, dose- and time-related inhibitory effect on the GJIC capacity of the cells . CONCLUSIONS: Even if further investigations are needed to better clarify the possible causal relationship between our findings, they indicate that: a) MDCK cells represent a suitable in vitro model for the study of Hg nephrotoxicity; b) GJIC function is, among those considered in our study, one of the most sensitive biological endpoints for investigating the mechanism(s) of Hg2+ specific toxicity.

Diabetes, 2002 Sep, 51(9), 2734 - 41
Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein; Nishizawa H et al.; Adiponectin, an adipose-specific secretory protein, exhibits antidiabetic and antiatherogenic properties . In the present study, we examined the effects of sex hormones on the regulation of adiponectin production . Plasma adiponectin concentrations were significantly lower in 442 men (age, 52.6 +/- 11.9 years {mean +/- SD}) than in 137 women (53.2 +/- 12.0 years) but not different between pre- and postmenopausal women . In mice, ovariectomy did not alter plasma adiponectin levels . In contrast, high levels of plasma adiponectin were found in castrated mice . Testosterone treatment reduced plasma adiponectin concentration in both sham-operated and castrated mice . In 3T3-L1 adipocytes, testosterone reduced adiponectin secretion into the culture media, using pulse-chase study . Castration-induced increase in plasma adiponectin was associated with a significant improvement of insulin sensitivity . Our results indicate that androgens decrease plasma adiponectin and that androgen-induced hypoadiponectinemia may be related to the high risks of insulin resistance and atherosclerosis in men.

Clin Sci (Lond), 2002 Aug, 103 Suppl 48, 94S - 97S
Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a; Kulathila R et al.; In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence . The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture . Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography . Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium . No interconversion of the two forms was detected after purification . Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA . However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively . These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1 . The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

Clin Sci (Lond), 2002 Aug, 103 Suppl 48, 35S - 38S
Three vasoactive peptides, endothelin-1, adrenomedullin and urotensin-II, in human tumour cell lines of different origin: expression and effects on proliferation; Takahashi K et al.; Evidence has accumulated showing that vasoactive peptides, such as endothelin-1, adrenomedullin and urotensin-II, are expressed in various kinds of tumour cells . In the present study, the expression of endothelin-1 and endothelin receptors was studied in eight human tumour cell lines: T98G (glioblastoma), IMR-32 and NB69 (neuroblastoma), BeWo (choriocarcinoma), SW-13 (adrenocortical carcinoma), DLD-1 (colonic carcinoma), HeLa (cervical carcinoma) and VMRC-RCW (renal carcinoma) . Reverse transcriptase-PCR showed expression of endothelin-1 mRNA in seven out of the eight cell lines, the exception being BeWo cells . ET(A) receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in the other cells . ET(B) receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but only weakly in T98G and HeLa cells . Immunoreactive endothelin was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 or BeWo cells . Treatment of T98G cells with an anti-endothelin-1 antibody or an anti-adrenomedullin antibody for 24 h decreased cell numbers to approx . 84% and 90% of control respectively . Treatment with the ET(A) receptor antagonist BQ-610 (1 microM) significantly decreased cell number to about 90% of control, whereas the ET(B) receptor antagonist BQ-788 had no significant effect . On the other hand, exogenously added endothelin-1, adrenomedullin or urotensin-II (0.1 microM) had no significant effects on cell number . These results suggest that endothelin-1 acts as a paracrine or autocrine growth stimulator in tumours . The effect of endothelin-1 on tumour growth appears to be mediated by the ET(A) receptor.

J Assist Reprod Genet, 2002 Aug, 19(8), 368 - 75
Objective assessments of temperature maintenance using in vitro culture techniques; Cooke S et al.; PURPOSE: To assess the ability of various facets of embryo culture (microscope stage warmers, volumes of culture media, culture vessel lids, and type of culture incubator) to maintain a constant temperature in vitro . METHODS: Ability to maintain 37.0 degrees C in the microenvironment of gametes was recorded by digital thermocouple in the chosen facets of in vitro culture . RESULTS: Stage warmers are highly variable in their ability to maintain the set temperature (range 33.8 degrees C-37.0 degrees C after 60 s) . Temperature loss in culture media is both volume and vessel dependent, and the direct heat transfer culture incubator (MINC) has superior temperature maintenance compared with a large volume air convection incubator (FORMA), where temperature regain from 35.0 degrees C to 37.0 degrees C took 5.5 min compared to >20 min . CONCLUSIONS: There are large measurable differences in the ability to maintain set temperature that depend on the stage warmer used, volume of media, use of vessel lids, and the type of incubator chosen for IVF culture.

J Bone Joint Surg Am, 2002 Aug, 84-A(8), 1405 - 12
Recombinant adeno-associated virus-mediated osteoprotegerin gene therapy inhibits wear debris-induced osteolysis; Ulrich-Vinther M et al.; BACKGROUND: Aseptic loosening of orthopaedic implants secondary to wear debris-induced osteolysis is a serious problem . Osteoprotegerin (OPG) is a natural decoy protein that inhibits osteoclast activation and bone resorption . This study investigated whether gene therapy using a recombinant adeno-associated viral vector that expresses OPG can inhibit wear debris-induced osteolysis . METHODS: A recombinant adeno-associated virus (rAAV) vector co-expressing OPG (rAAV-OPG-IRES-EGFP) was generated . A control vector expressing b-galactosidase (rAAV-LacZ) was also prepared . In vitro validation experiments were performed to determine rAAV-OPG-IRES-EGFP transduction efficiency, OPG expression level and function in bone wafer, and osteoclastic activity . The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear debris-induced osteolysis was then evaluated in a mouse calvarial model in which a single intramuscular injection of the vector was administered prior to the introduction of the wear debris . The effects of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis and bone resorption were determined by histomorphometry on day 10 . RESULTS: In vitro experiments revealed that 100% of human embryonic kidney 293 cells were transduced at a multiplicity of infection of 1000 with both rAAV-OPG-IRES-EGFP and rAAV-LacZ . At a rAAV-OPG-IRES-EGFP multiplicity of infection of 1000, an OPG concentration of 135 ng/mL of culture media was achieved after four days . Using a bone-wafer assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP reduced resorption sevenfold compared with parathyroid hormone-stimulated controls and elevenfold compared with rAAV-LacZ controls . Furthermore, a seventeenfold decrease in RANKL and macrophage colony-stimulating factor-induced splenocyte osteoclastogenesis was observed in co-cultures containing rAAV-OPG-IRES-EGFP-infected fibroblasts . In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable transduction of myocytes at the injection site and a significant increase in expression of serum OPG levels by the second day (p < 0.05) . Maximal concentrations were obtained on day 6 and then leveled off throughout the observation period . In contrast, serum OPG could not be detected in the sham-treated, uninfected titanium-stimulated, or rAAV-LacZ-infected mice . In the control mice, titanium implantation resulted in a threefold increase in the mean number of osteoclasts adjacent to the sagittal suture as well as a twofold increase in the mean area of soft tissue in the sagittal suture compared with the sham-treated mice . In contrast, osteoclast numbers remained at basal levels, and the area of soft tissue in the sagittal suture was markedly reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment, demonstrating a complete inhibition of osteolysis in response to titanium particles . CONCLUSIONS: A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector can efficiently transduce myocytes to produce high levels of OPG . The OPG effectively inhibits wear debris-induced osteoclastogenesis and osteolysis . Clinical Relevance: Currently, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis . Although preclinical studies have identified potential drug therapies (i.e., bisphosphonates), there is no evidence that these drugs can effectively treat aseptic loosening in patients . This is the first evidence that in vivo OPG gene therapy can be used to prevent wear debris-induced osteolysis.

Anticancer Res, 2002 Jul-Aug, 22(4), 2423 - 7
Hyaluronidase is more elevated in human brain metastases than in primary brain tumours; Delpech B et al.; BACKGROUND: Hyaluronidase is hypothesised to play a role in cancer invasion and metastasis formation . MATERIALS AND METHODS: Hyaluronidase activity was investigated at pH 3.8 in extracts of 30 human brain tumours (17 glioblastomas and 13 brain metastases of carcinomas) and in cancer cell cultures with the ELSA method and zymography . RESULTS: In brain metastases, hyaluronidase activities were significantly higher than in glioma extracts (9.16 +/- 4.48 mU/g vs 4.25 +/- 5.74) which was not explained by serum hyaluronidase contamination . Serum hyaluronidase of tumour patients' sera was within the normal values determined in 28 matched blood donors'sera (33.8 +/- 11 U/l) . The maximum hyaluronidase/albumin (U/g) ratio was 0.9, below which the hyaluronidase content of tumours was below the maximum value calculated from the albumin content of the tumour extract and could not be considered as local production by tumour cells . The hyaluronidase content and hyaluronidase/albumin ratio of metastasis extracts was significantly higher than in glioma extracts and patients' sera, whereas no significant difference was found between the ratios of glioma extracts and sera . The production of hyaluronidase was studied in cell extracts and in culture media of 3 human glioma-derived cell lines and of the brain metastasis-derived cell line SA87 . Hyaluronidase activity of the metastasis-derived cell line SA87 was 100 to 1000-fold that of glioma cell lines . CONCLUSION: These results suggest that hyaluronidase is associated with the more aggressive cancer cells and is directly or indirectly involved in brain metastasis phenotype.

Quintessence Int, 2002 Jul-Aug, 33(7), 496 - 502
Root instrumentation with an erbium:yttrium-aluminum-garnet laser: effect on the morphology of fibroblasts; Rossa CJ et al.; OBJECTIVE: The purpose of this study was to evaluate the effect of erbium:yttrium-aluminum-garnet laser instrumentation of root surfaces on the morphology of fibroblasts from continuous lineage . METHOD AND MATERIALS: Dentinal slices with 4 mm2 of surface area were obtained from teeth extracted for severe periodontal involvement . Specimens were assigned to one of three treatment groups: group 1, application of the laser with an energy level of 250 mJ at 103 pulses per second; group 2, application of the laser with an energy level of 80 mJ at 166 pulses per second; and group 3, similar to group 2, but with concomitant water irrigation of the device . The specimens were incubated in multiwell plates containing cell culture media . After 24 hours, the specimens were submitted to routine preparation for scanning electron microscopy . Three independent and blind examiners used photomicrographs to evaluate the morphology of the fibroblasts: 0 = without cells; 1 = flat cells; 2 = round cells; and 3 = combination of round and flat cells . RESULTS: Statistical analysis indicated that there were significant differences among treatment groups and that group 3 was significantly different from groups 1 and 2 . CONCLUSION: There was no difference between groups 1 and 2 in the morphology of fibroblasts . Laser instrumentation with concomitant irrigation impaired the adhesion of fibroblasts to dentinal surfaces.

Life Sci, 2002 Apr 21, 70(21), 2521 - 33
Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function; Pratt PF et al.; During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer . During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated . Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs . Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence . The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs . When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence . However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence . Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs . We examined the effects of the EETs on PMN aggregation . 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal . 5,6-, 11,2- and 14,15-EETs also stimulated aggregation . FMLP stimulated the production of superoxide; however, 8,9-EET did not . These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation . Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury.

Cancer Biol Ther, 2002 Mar-Apr, 1(2), 168 - 76
Pharmocologic inhibitors of the mitogen activated protein kinase cascade have the potential to interact with ionizing radiation exposure to induce cell death in carcinoma cells by multiple mechanisms; Qiao L et al.; Recent studies have shown that inhibition of stress-induced signaling via the mitogen activated protein kinase (MAPK) pathway can potentiate the toxic effects of chemotherapeutic drugs and ionizing radiation . Because of these observations, we have further investigated the impact upon growth and survival of mammary (MDA-MB-231, MCF7, T47D), prostate (DU145, LNCaP, PC3) and squamous (A431) carcinoma cells following irradiation and combined long-term exposure to MEK1/2 inhibitors . Exposure of carcinoma cells to ionizing radiation resulted in MAPK pathway activation initially (0-4 h) and modestly enhanced MAPK activity at later times (24 h-96 h) . Inhibition of radiation-induced MAPK activation using MEK1/2 inhibitors potentiated radiation-induced apoptosis in two waves, at 21-30 h and 96-144 h after exposure . The potentiation of apoptosis was not observed in MCF7, LNCaP, or PC3 cells . At 24 h, the potentiation of apoptosis was independent of radiation dose whereas at 108 h, apoptosis correlated with increasing dose . Removal of the MEK1/2 inhibitor either 6 h or 12 h after exposure abolished the potentiation of apoptosis at 24 h . At this time, the potentiation of apoptosis correlated with cleavage of pro-caspases -8, -9 and -3, and with release of cytochrome c into the cytosol . Inhibition of caspase function using a pan-caspase inhibitor ZVAD blocked the enhanced apoptotic response at 24 h . Selective inhibition of caspase 9 with LEHD or caspase 8 with IETD partially blunted the apoptotic response in MDA-MB-231, DU145 and A431 cells, whereas inhibition of both caspases reduced the response by > 90% . Removal of the MEK1/2 inhibitor either 24 h or 48 h after exposure abolished the potentiation of apoptosis at 108 h . Incubation of cells with ZVAD for 108 h also abolished the potentiation of apoptosis . In general agreement with the finding that prolonged inhibition of MEK1/2 was required to enhance radiation-induced apoptosis at 108 h, omission of MEK1/2 inhibitor from the culture media during assessment of clonogenic survival resulted in either little or no significant alteration in radiosensitivity . Collectively, our data show that combined exposure to radiation and MEK1/2 inhibitors can reduce survival in some, but not all, tumor cell types . Prolonged blunting of MAPK pathway function following radiation exposure is required for MEK1/2 inhibitors to have any effect on carcinoma cell radiosensitivity.

J Vasc Surg, 2002 Aug, 36(2), 263 - 70
Effect of adenoviral titer and instillation pressure on gene transfer efficiency to arterial and venous grafts ex-vivo; Brevetti LS et al.; OBJECTIVE: Adenoviral-mediated gene transfer to arterial and venous grafts has potential in the treatment of a number of vascular diseases . Despite widespread use of these vectors to mediate gene transfer to blood vessel walls, the optimal transduction conditions for each type of vessel has yet to be determined . Our objective was to study the effect of adenoviral titer and instillation pressure on efficiency of gene transfer to arterial and venous grafts ex-vivo . METHODS: Jugular vein and carotid artery segments of 8 cm were harvested from Yorkshire Cross pigs . Tissue culture media or different titers of an adenoviral vector encoding human placental alkaline phosphatase (hpAP) were instilled into venous and arterial grafts at 0 mm Hg or 80 to 100 mm Hg of pressure and bathed externally in the same solution at 37 degrees C for 30 minutes . The grafts were rinsed, opened longitudinally, and incubated in culture media at 37 degrees C for 48 hours . Grafts were fixed and stained for hpAP transgene expression to quantitate percent luminal transduction or homogenized for alkaline phosphatase (AP) activity to determine total transmural transduction . RESULTS: For venous grafts, the percent luminal area stained for hpAP was greatest with 10(8) plaque-forming units/mL at 0 mm Hg (81% +/- 7%) and decreased with increasing titers (53% +/- 9% at 10(9) pfu/mL and 44% +/- 11% at 5 x 10(9) pfu/mL; n = 7; P <.05) . No increase in percent luminal area stain was achieved with an instillation pressure of 80 to 100 mm Hg at any viral titer . The inverse finding was observed in arterial grafts . For arterial grafts, the greatest percent luminal area stained was achieved with 5 x 10(9) pfu/mL at 80 to 100 mm Hg (76% +/- 7%) . An instillation pressure of 80 to 100 mm Hg increased the percent luminal area stained at 10(8) pfu/mL from 31% +/- 9% to 66% +/- 8% (n = 8; P =.01) . For venous grafts, total AP activity peaked with 10(9) pfu/mL at 0 mm Hg and decreased with an instillation pressure of 80 to 100 mm Hg (30.6 +/- 9.7 U/mg versus 10.9 +/- 2.5 U/mg; n = 7; P <.01) . However, for arterial grafts, total AP activity peaked with 5 x 10(9) pfu/mL (0 mm Hg) and increased with an instillation pressure of 80 to 100 mm Hg (32.8 +/- 9.9 U/mg versus 63.4 +/- 20.5 U/mg; n = 8; P <.05) . CONCLUSION: High transduction efficiency can be achieved with adenoviral-mediated gene transfer of arterial and venous grafts . Gene transfer with the vascular graft's physiologic pressure conditions improved transduction efficiency for the artery (80 to 100 mm Hg) and vein (0 mm Hg) . Comprehensive analysis of adenoviral transduction conditions is important to realize the full promise of adenoviral-mediated gene transfer.

Clin Exp Immunol, 2002 Aug, 129(2), 379 - 84
Functional changes in rheumatoid fibroblast-like synovial cells through activation of peroxisome proliferator-activated receptor gamma-mediated signalling pathway; Yamasaki S et al.; Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand dependent transcriptional factor known to be a regulator of adipogenesis . Recent studies have also shown that stimulation of PPARgamma inhibits the transcriptional activities of other nuclear factors and down-regulates proinflammatory cytokine synthesis in T cells and monocytes . We examined, in the present study, the functional significance of PPARgamma expressed in fibroblast-like synovial cells (FLS) isolated from patients with rheumatoid arthritis (RA) . Incubation of FLS with a synthetic PPARgamma ligand, troglitazone, inhibited endogenous production of TNF-alpha, IL-6 and IL-8, as well as matrix metalloprotease-3 (MMP-3), without inducing apoptosis of the cells . The gelatinase activity of FLS culture media was also inhibited by troglitazone . Electrophoretic mobility shift assay (EMSA) showed a significant reduction in the DNA binding activity of NF-kappaB in troglitazone-treated FLS in response to TNF-alpha or IL-1beta . Moreover, long-term cultivation of FLS with troglitazone resulted in morphological changes with marked lipid accumulation in these cells . Our results show a negative regulatory function for PPARgamma on cytokine and MMP production together with inhibition of cytokine-mediated inflammatory responses in rheumatoid synovial cells . Our results also suggest that FLS could differentiate into adipocyte-like cells in the presence of proper stimulatory signals including PPARgamma.

Biomaterials, 2002 Oct, 23(19), 4001 - 10
An evaluation of accelerated Portland cement as a restorative material; Abdullah D et al.; Biocompatibility of two variants of accelerated Portland cement (APC) were investigated in vitro by observing the cytomorphology of SaOS-2 osteosarcoma cells in the presence of test materials and the effect of these materials on the expression of markers of bone remodelling . Glass ionomer cement (GIC), mineral trioxide aggregate (MTA) and unmodified Portland cement (RC) were used for comparison . A direct contact assay was undertaken in four samples of each test material, collected at 12, 24, 48 and 72 h . Cell morphology was observed using scanning electron microscopy (SEM) and scored . Culture media were collected for cytokine quantification using enzyme-linked immunosorbent assay (ELISA) . On SEM evaluation, healthy SaOS-2 cells were found adhering onto the surfaces of APC variant, RC and MTA . In contrast, rounded and dying cells were observed on GIC . Using ELISA, levels of interleukin (IL)-1beta, IL-6, IL-18 and OC were significantly higher in APC variants compared with controls and GIC (p<0.01), but these levels of cytokines were not statistically significant compared with MTA . The results of this study provide evidence that both APC variants are non-toxic and may have potential to promote bone healing . Further development of APC is indicated to produce a viable dental restorative material and possibly a material for orthopaedic

J Clin Endocrinol Metab, 2002 Aug, 87(8), 3774 - 8
Estrogen represses whereas the estrogen-antagonist ICI 182780 stimulates placental CRH gene expression; Ni X et al.; CRH and estrogens, produced by placental trophoblasts, have been suggested to play pivotal roles in the control of human parturition . Estrogen has been shown to affect hypothalamic CRH expression . Therefore, we evaluated 17 beta-estradiol (E2) in the regulation of CRH gene expression in placental cells . E2 inhibited CRH mRNA expression in a dose-dependent manner, which paralleled the decrease in CRH protein levels in culture media . A complete estrogen receptor (ER) antagonist, ICI 182780, not only blocked repression of CRH mRNA levels by E2, but up-regulated CRH mRNA and protein synthesis . An ER alpha-mixed agonist/antagonist and ER beta antagonist, 4-hydroxytamoxifen, also down-regulated CRH gene expression . Using quantitative RT-PCR, we found that placental trophoblasts express predominantly the ER alpha form of the receptor . Transient transfection assays conducted in the choriocarcinoma cell line JEG-3 demonstrated that E2 repressed CRH promoter activity, whereas the antagonist ICI 182780 up-regulated CRH promoter activity when ER alpha was cotransfected . These studies demonstrate that E2 represses placental CRH gene expression through an ER alpha-mediated mechanism . Estrogen may therefore modulate placental CRH production, influencing the rate of rise of maternal plasma CRH concentrations and potentially the length of gestation.

Diagn Microbiol Infect Dis, 2002 Aug, 43(4), 297 - 302
Rapid differentiation between clinically relevant mycobacteria in microscopy positive clinical specimens and mycobacterial isolates by line probe assay; Johansen IS et al.; The Inno LiPA Mycobacteria assay, based on PCR amplification of the 16-23S rRNA spacer region of Mycobacterium species, has been designed for identification of mycobacteria grown in culture media and discrimination between Mycobacterium tuberculosis complex, M . avium, M . intracellulare, M . kansasii, M . gordonae, M . xenopi, scrofulaceum and M . chelonae group including M . abscessus . In order to evaluate the system as a fast diagnostic tool, the assay was for the first time used directly on 14 microscopy positive clinical specimens and 71 isolates and the results were compared to those of conventional identification using 16S rDNA analysis and biochemical properties . The assay only misidentified one strain, which was found to be M . avium complex instead of M . intracellulare as found by the conventional tests . The assay allows rapid discrimination of the eight most clinically relevant mycobacteria in microscopy positive clinical specimens and isolates within 6 h in the same procedural run.

Tsitologiia, 2002, 44(4), 357 - 63
{Properties of endoribonuclease activity of proteasomes from K562 cells . II . Analysis of specific mRNA nucleolysis by proteasomes}; Mittenberg AG et al.; The ability of 26S proteasomes from the human proerythroleukaemic cell line K562 to degrade high-molecular-weight cytoplasmic RNAs, particularly specific messenger RNA, has been detected . The addition of hemin to K562 cells in the culture media leads to redistribution of proteasomes and their migration mainly to the cytoplasm . The human wild type p53 gene mRNA was shown to be specifically nucleolized by proteasomes . These particles displayed endoribonuclease activity towards mRNA for Renilla sp . luciferase . Proteasomes also specifically degraded Alu-containing mRNAs . A supposition is made about the involvement of proteasomes in stability control of specific RNA groups.

Toxicol Appl Pharmacol, 2002 Jul 15, 182(2), 160 - 8
Percutaneous absorption of explosives and related compounds: an empirical model of bioavailability of organic nitro compounds from soil; Reifenrath WG et al.; The percutaneous absorption potentials of (14)C-labeled 2,4,6-trinitrotoluene (TNT), trinitrobenzene, 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 2,6-diamino-4-nitrotoluene, N-methyl-N,2,4,6-tetranitrobenzamine, hexahydro-1,3,5-trinitro-1,3,5-triazine, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, and 2,2-thiobis(ethanol) were determined from two soil types, Yolo having 1.9% carbon and Tinker having 9.5% carbon . TNT skin absorption from another low-carbon soil (Umatilla) was also determined . Approximately 10 microg/cm(2) of radiolabeled compound was applied in 5 microl of acetone or 10 mg/cm(2) of soil to excised pigskin mounted in skin penetration-evaporation chambers . Absorption from acetone served as a control . Radiolabel recovered from the dermis and tissue culture media (receptor fluid) was summed to determine the percentage of absorption from the soils . For each compound, percentage absorptions of radiolabel were highest from acetone solution and lowest from Tinker soil, with the results from Yolo soil being intermediate . Skin absorptions of TNT from Yolo and Umatilla soils were similar . For TNT in all vehicles, the penetration rate of radiolabel into the receptor fluid was highest during the 1- to 2-h interval after dosing . HPLC analysis of TNT radiolabel in receptor fluid at maximum flux indicated extensive conversion to monoamino derivatives and other metabolites . For 2,4-DNT and 2,6-DNT applications in Yolo soil, percentage absorptions approached those obtained from acetone applications . After 2,4-DNT and 2,6-DNT applications (acetone and soils), HPLC analysis of radiolabel in receptor fluid during the period of maximum flux revealed no significant metabolites . Skin absorption of the nitro compounds from soils was found to correlate with the compound's water solubility and vapor pressure . These findings formed the basis of an empirical model to predict skin bioavailability.

Pancreatology, 2002, 2(4), 402 - 6
Specific amino acid profile in culture media conditioned by human pancreatic cancer cell lines; Wang F et al.; BACKGROUND: In malignant diseases, circulating amino acid profiles correlate with organ sites of malignancy . Direct effects of malignant cells on the extracellular amino acid profile are still uncertain . METHODS: Free amino acids were measured in serum-free culture media (RPMI-1640) conditioned by two human pancreatic cancer cell lines (Panc-1 and HPAF), a hamster pancreatic cancer cell line (PC-1), a human epidermoid carcinoma cell line (A-431), and a human fibroblastic cell line (Ag-1523) . Non-conditioned RPMI-1640 medium was used as control . RESULTS: Amino acid profiles were changed in all the conditioned media, caused by a decrease or increase in the original amino acids and by the appearance of amino acids that were not present in non-conditioned medium . Media conditioned by two human pancreatic cancer cell lines showed similar amino acid profiles, which were characterized by a decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine . CONCLUSION: Culture media show changed amino acid profiles following incubation with cell lines of pancreatic or non-pancreatic origins . Different human pancreatic cancer cell lines cause similar changes in amino acid profiles of media .

J Biol Chem, 2002 Oct 11, 277(41), 38148 - 58 Epub 2002 Jul 22.
Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex . Evidence that all strains synthesize glycosylated p-hydroxybenzoic methly esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene; Constant P et al.; Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans . They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated . Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M . tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains . To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M . tuberculosis were analyzed . We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M . tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor . We also show that all the strains of M . tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M . leprae, and strains of M . tuberculosis that produce phenolphthiocerol derivatives . Complementation of the H37Rv strain of M . tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M . bovis BCG restored phenolphthiocerol glycolipids production . Conversely, disruption of the pks15/1 gene in M . bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid . These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.

Anim Reprod Sci, 2002 Aug 15, 72(3-4), 137 - 51
Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers; Reis A et al.; To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals . More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M . = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%) . The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M . = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively) . Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ . Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05) . In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1 . Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones . Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process .

Hypertens Res, 2002 May, 25(3), 455 - 60
Lowering of blood pressure improves endothelial dysfunction by increase of nitric oxide production in hypertensive rats; Hatta T et al.; To investigate the effects of angiotensin converting enzyme inhibitor (ACE-I) and other antihypertensive agents on the nitric oxide (NO) release during hypertension, seven- and fourteen-week-old SHR and deoxycorticosterone acetate (DOCA)-salt rats were treated with hydralazine, manidipine (Ca antagonist) or quinapril (ACE-I) for 3 weeks to lower blood pressure . Systolic blood pressure (SBP) was measured by the tail cuff method once each week . Endothelial cells (ECs) derived from the descending aorta of the treated rats were cultured and NOx levels in culture media were measured with an NO analyzer based on the Griess reaction . In both SHR and DOCA-salt rats, antihypertensive therapy lowered SBP to levels similar to those of control rats . The only exception was quinapril treatment of DOCA-salt rats . Although NOx release by ECs derived from hypertensive rats was improved by antihypertensive therapy, the effect was most pronounced in SHR treated with quinapril . In addition, restoration of NOx release was much more remarkable in younger SHR . NOx release was significantly higher in DOCA-salt rats treated with quinapril than in control rats without reduction of SBP . These results suggest that lowering blood pressure improves release of NO by ECs during hypertension and that the time at which antihypertensive therapy is started is also important to preserve endothelial function . Furthermore, ACE-I is suggested to protect endothelial function by increasing NO production in addition to lowering blood pressure.

J Hand Surg {Am}, 2002 Jul, 27(4), 615 - 20
Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production; Klein MB et al.; Flexor tendon healing is complicated by adhesions to the surrounding sheath . Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing . We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production . Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately . Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3 . Cell number and collagen I and III production were measured and compared with unsupplemented control cultures . The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance . There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms . Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.

Endocrinology, 2002 Aug, 143(8), 2872 - 9
Antigen presentation by vaginal cells: role of TGFbeta as a mediator of estradiol inhibition of antigen presentation; Wira CR et al.; Estradiol is known to inhibit antigen presentation in the vagina . We report here that TGFbeta mediates the action of estradiol on vaginal antigen presenting cells (APC) . When vaginal APC from ovariectomized rats were incubated with increasing concentrations of TGFbeta1 and TGFbeta2 in the presence of ovalbumin-specific T cells and ovalbumin, both TGFbeta1 and TGFbeta2 inhibited vaginal cell antigen presentation, whereas IL-6, IL-10, and TNFalpha had no consistent effect . In other experiments, estradiol-induced inhibition of antigen presentation by vaginal cells was partially reversed when vaginal APC were incubated with anti-TGFbeta antibody . In contrast, anti-TNFalpha, anti-IL-6, and anti-IL-10 had no effect on antigen presentation . The effect of anti-TGFbeta was seen with vaginal APC from ovariectomized rats treated with estradiol for 1 d as well as 3 d . Finally, analysis of TGFbeta in the culture media of vaginal cells from saline- and estradiol-treated rats indicated that the TGFbeta produced is biologically active . In response to estradiol, vaginal cell production of TGFbeta was significantly greater than that seen with control cells . These studies suggest that estradiol regulation of antigen presentation by vaginal cells is mediated through the local production of TGFbeta by vaginal cells.

Int J Radiat Oncol Biol Phys, 2002 Aug 1, 53(5), 1314 - 8
Pronounced radiosensitization of cultured human cancer cells by COX inhibitor under acidic microenvironment; Shah T et al.; PURPOSE: To demonstrate the influence of pH on the cytotoxicity and radiosensitization by COX (cyclooxygenase) -1 and -2 inhibitors using established human cancer cells in culture . METHODS AND MATERIALS: Nonselective COX inhibitor, ibuprofen (IB), and selective COX-2 inhibitor, SC-236, were used to determine the cytotoxicity and radiosensitization at varying pH of culture media . Human colon carcinoma cell line (HT-29) was exposed to the drug alone and in combination with radiation at different pH of the cell culture media . The end point was clonogenic ability of the single-plated cells after the treatment . RESULTS: Cytotoxicity and radiosensitization of IB increased with higher drug concentration and longer exposure time . The most significant radiosensitization was seen with IB (1.5 mM) for 2-h treatment at pH 6.7 before irradiation . The dose-modifying factor as defined by the ratio of radiation doses required to achieve the same effect on cell survival was 1.8 at 10% survival level . In contrast, SC-236 (50 microM for 2-8 h) showed no pH-dependent cytotoxicity . There was modest increase in the cell killing at lower doses of radiation . CONCLUSION: An acidic pH was an important factor affecting the increased cytotoxicity and radiosensitization by ibuprofen . Radiation response was enhanced at shoulder portion of the cell survival curve by selective COX-2 inhibitor.

Eur J Vasc Endovasc Surg, 2002 Jul, 24(1), 72 - 80
Enhanced invasive properties exhibited by smooth muscle cells are associated with elevated production of MMP-2 in patients with aortic aneurysms; Goodall S et al.; BACKGROUND: abdominal aortic aneurysms (AAA) are associated with excessive vascular matrix remodelling . Recent findings suggest a systemic overproduction of matrix metalloproteinases-2 (MMP-2) by vascular smooth muscle cells (SMC) may be pivotal aetiologically . SMC migration is facilitated by MMP mediated proteolysis of the basement membrane and extracellular matrix . Our aim was to see if enhanced MMP-2 production by these SMC exhibit increased invasion, in an in vitro model of migration . METHOD: SMC were derived from inferior mesenteric vein (IMV) harvested from patients undergoing aneurysm repair (n=6) or colectomy for diverticulosis (n=6, control) . Using a modified Boyden chamber chemotaxis was measured towards platelet derived growth factor (PDGF) and foetal calf serum (FCS) and invasion through a Matrigel layer . MMP-2 production was quantified by ELISA and gelatin zymography . RESULTS: chemoattractant studies demonstrated no difference in the effect of PDGF or FCS between the two populations of SMC . However, invasive studies demonstrated a significant increase in the number of migrating SMC isolated from IMV of AAA patients . Analysis of culture media extracts revealed that this difference was associated with a significant increase in production of MMP-2 . CONCLUSION: SMC derived from patients with AAA demonstrate increased invasive properties when compared to a control group . Increased migration appears to be due to overproduction of MMP-2 . The enhanced migratory potential of these SMC may lead to extracellular matrix remodelling and subsequent medial disruption demonstrated in the aneurysmal aorta . These data further support evidence of the proteolytic role of MMP-2 in cell migration.

J Neuropathol Exp Neurol, 2002 Jul, 61(7), 585 - 96
Galectin-1 modulates human glioblastoma cell migration into the brain through modifications to the actin cytoskeleton and levels of expression of small GTPases; Camby I et al.; We show that high-grade astrocytic tumors with high levels of galectin-1 expression are associated with dismal prognoses . The immunohistochemical analysis of galectin-1 expression of human U87 and U373 glioblastoma xenografts from the brains of nude mice revealed a higher level of galectin-1 expression in invasive areas rather than non-invasive areas of the xenografts . Nude mice intracranially grafted with U87 or U373 cells constitutively expressing low levels of galectin-1 (by stable transfection of an expression vector containing the antisense mRNA of galectin-1) had longer survival periods than those grafted with U87 or U373 cells expressing normal levels of galectin-1 . Galectin-1 added to the culture media markedly and specifically increased cell motility levels in human neoplastic astrocytes . These effects are related to marked modifications in the organization of the actin cytoskeleton and the increase in small GTPase RhoA expression . All the data obtained indicate that galectin-1 enhances the migratory capabilities of tumor astrocytes and, therefore, their biological aggressiveness.

J Neurochem, 2002 Jul, 82(2), 240 - 8
An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells; Nunez-Millacura C et al.; Intracellular reactive iron is a source of free radicals and a possible cause of cell damage . In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons . Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool . The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant . When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil . In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased . IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake . The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine . Culturing cells with iron resulted in proteins and DNA modifications . In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media . As a consequence, iron accumulated and generated oxidative damage . This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.

Biochem Pharmacol, 2002 Jul 15, 64(2), 267 - 76
Novel mechanism of Vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes; Andres D et al.; Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases . It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation which are directly involved in the CsA hepatotoxicity . As antioxidant, Vitamin E (VitE) has been used to diminish the toxicity of CsA in vitro . Besides its direct action as the classical antioxidant implicated in preventing lipid peroxidation, we decided to investigate the effect of VitE on the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD) catalase and glutathione peroxidase (GPx) on CsA cytotoxicity in primary cultures of rat hepatocytes . In cells incubated in the presence of CsA, there was an increase in the expression and activity of MnSOD and CuZnSOD but not in that of catalase and GPx . However, when hepatocytes were coincubated with CsA and VitE, an increase in the expression and activity in all antioxidant enzymes (MnSOD, CuZnSOD, catalase and GPx) was observed . In conclusion, we suggest (a) that the imbalance between SOD and catalase/GPx by the effect of CsA is the main mechanism responsible for peroxide accumulation and cell death in hepatocytes, and (b) that the presence of VitE in culture media reduces the oxidative stress through the inhibition of lipid peroxidation, but also through the increase of the expression and activity of catalase and GPx which allows the restoration of SOD and catalase/GPx coordination, indispensable for the correct cell defense against ROS.

Med Sci Monit, 2002 Jul, 8(7), BR248 - 53
Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production; Dillon BJ et al.; BACKGROUND: Nitric oxide (NO) is a biomediator believed to be synthesized primarily from extracellular arginine . Various methodologies have been used to inhibit NO synthesis so as to elucidate its physiological and pathophysiological functions . Several investigators have utilized various argin ine degrading enzymes as a means of lowering extracellular arginine . Arginase, most commonly derived from mammalian sources, has been most often used . However, arginase has failed to inhibit NO synthesis . Therefore, a systematic biochemical characterization of arginase and arginine deiminase (ADI) derived from M . Hominus was undertaken . MATERIAL/METHODS: The murine macrophage cell line N-9 was treated with either arginase or arginine deiminase to determine the effect on intracellular and extracellular arginine and nitric oxide production . RESULTS: Arginase was found to have an alkaline pH optima(approximately 9.5) with little enzyme activity at physiological pH . In contrast, the pH optima of ADI was approximately 6.5, retaining >70% of its activity at physiological pH . ADI had more than 1000 fold higher affinity for arginine (Km approximately 30 KM for ADI vs approximately 45 mM for arginase), and was able to lower arginine levels to a much greater extent than arginase . ADI, unlike arginase, was effective in lowering extracellular arginine in tissue culture media and inhibit NO production by the murine macrophage cell line N-9 in response to gamma interferon and LPS stimulation . CONCLUSIONS: These data suggest that ADI may be useful for delineating the role of NO in a variety of biological systems as well as determining the role of extracellular arginine in its synthesis.

Equine Vet J, 2002 Jul, 34(4), 378 - 82
Equine oocyte maturation with epidermal growth factor; Lorenzo PL et al.; Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species . This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods . Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group (TCM 199 supplemented with 10% v/v oestrous mare serum) . Each group was divided further into 3 treatments with tyrphostin A-47, a specific tyrosine kinase inhibitor, at 0, 10(-4) and 10(-6) mmol/l . Maturation was determined as the percentage of oocytes reaching metaphase II stage at the end of the culture period . Immunohistochemical detection of EGF-receptor (EGFR) was performed using a streptoavidin-biotin method . The recovery rate and oocyte retrieval were 84.6% (recovered oocytes/follicles aspirated) and 6.55 (oocytes/mare), respectively . Treatment with EGF significantly (P<0.05) increased the incidence of metaphase II stage compared with the control group (69.4 vs . 26.9% in controls, respectively) . The specific-tyrosine kinase inhibitor A-47 was effective in suppressing EGF-effect on EGF-cultured oocytes; no significant differences were observed in EMS-supplemented oocytes when cultured with A-47 . EGF-receptor was localised in follicles, with localisation being more prominent in the cumulus than in mural granulosa cells . This finding, together with the increase of oocyte nuclear maturation rate when using EGF in culture media and the inhibition of maturation by tyrphostin A-47, suggests a physiological role for EGF in the regulation of equine oocyte maturation . The results should help successful development of assisted reproductive technology in the horse.

J Biomed Mater Res, 2002 Sep 15, 61(4), 619 - 27
The effect of polyethylene particle phagocytosis on the viability of mature human macrophages; Xing S et al.; Macrophages are the major cell type observed in the inflammatory membrane retrieved at implant revision surgery . In this study, mature human monocyte-derived macrophages (MDM) were adapted to a previously established in vitro model to examine the influence of high-density polyethylene (HDPE) particulate (4-10 microm) on MDM viability . HDPE particles were suspended in soluble type I collagen, which subsequently was solidified on glass coverslips . Mature human macrophages, derived from differentiating peripheral blood monocytes on polystyrene for 10 days, were incubated in culture media on collagen controls and collagen-particle substrata for 31 days . Histologic analysis demonstrated that MDMs were in contact with the particles at 2 h . The majority of the particles were associated with the cells within 24 h . Based on electron microscopy, those cells associated with the particles appeared to be morphologically activated rather than necrotic or apoptotic . Assessment of cell viability revealed no differences among the groups at 24 h, but at 31 days significantly more viable cells and higher DNA values were found associated with the particle groups versus the collagen controls . The histologic results validate human mature MDMs as a clinically relevant cell type for study of the role of polyethylene particulate in aseptic loosening . The cell viability results indicate that phagocytosis of HDPE is not toxic to MDMs but in fact prolongs MDM survival . The long-lived MDMs may play a role in perpetuating chronic inflammation surrounding implants .

Endocr Pathol, 1995 Summer, 6(2), 125 - 134
Basic Fibroblast Growth Factor Expression by Two Prolactin and Thyrotropin-Producing Pituitary Adenomas; Ezzat S et al.; We investigated the expression of basic fibroblast growth factor (bFGF) in an aggressive type of PRL and TSH-producing pituitary adenoma . Immunocytochemistry and electron microscopy were used to characterize the tumors removed from two patients . lmmunoassays were used to measure hormone and bFGF levels in vitro and in vivo . Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect bFGF mRNA expression by these tumors . Morphologically, these tumors were characterized by an unusual plurihormonal pattern with expression of PRL and TSH and the ultrastructural characteristics of the silent subtype 3 adenoma; in addition, both adenomas displayed marked interstitial fibrosis . bFGF was measurable in the circulation of these patients ranging from 7.5-20.5 pg/mL (normal < 1 pg/mL) . bFGF concentrations were reduced following surgical adenomectomy . bFCF in culture media was present in concentrations of 197-387 pg/24h/10(5) cells . bFGF mRNA expression was identified in both adenomas examined . bFGF levels were unaltered in the culture media and in the serum of patients following GnRH and TRH treatment . In conclusion, the expression of bFGF by these plurihormonal pituitary adenomas suggests the possibility that it may play a role in the development of fibrosis and tumor cell proliferation of this unusual type of pituitary neoplasm.

Rapid Commun Mass Spectrom, 2002, 16(14), 1389 - 97
Inverse 15N-metabolic labeling/mass spectrometry for comparative proteomics and rapid identification of protein markers/targets; Wang YK et al.; The inverse labeling/mass spectrometry strategy has been applied to protein metabolic (15)N labeling for gel-free proteomics to achieve the rapid identification of protein markers/targets . Inverse labeling involves culturing both the perturbed (by disease or by a drug treatment) and control samples each in two separate pools of normal and (15)N-enriched culture media such that four pools are produced as opposed to two in a conventional labeling approach . The inverse labeling is then achieved by combining the normal (14)N-control with the (15)N-perturbed sample, and the (15)N-control with the (14)N-perturbed sample . Both mixtures are then proteolyzed and analyzed by mass spectrometry (coupled with on-line or off-line separation) . Inverse labeling overcomes difficulties associated with protein metabolic labeling with regard to isotopic peak correlation and data interpretation in the single-experiment approach (due to the non-predictable/variable mass difference) . When two data sets from inverse labeling are compared, proteins of differential expression are readily recognized by a characteristic inverse labeling pattern or apparent qualitative mass shifts between the two inverse labeling analyses . MS/MS fragmentation data provide further confirmation and are subsequently used to search protein databases for protein identification . The methodology has been applied successfully to two model systems in this study . Utilizing the inverse labeling strategy, one can use any mass spectrometer of standard unit resolution, and acquire only the minimum, essential data to achieve the rapid and unambiguous identification of differentially expressed protein markers/targets . The strategy permits quick focus on the signals of differentially expressed proteins . It eliminates the detection ambiguities caused by the dynamic range of detection . Finally, inverse labeling enables the detection of covalent changes of proteins responding to a perturbation that one might fail to distinguish with a conventional labeling experiment .

J Neurosci Res, 2002 Jul 1, 69(1), 94 - 103
Cytokines, chemokines, and cytokine receptors in human microglia; Lee YB et al.; Enriched populations of human microglial cells were isolated from mixed cell cultures prepared from embryonic human telencephalon tissues . Human microglial cells exhibited cell type-specific antigens for macrophage-microglia lineage cells including CD11b (Mac-1), CD68, B7-2 (CD86), HLA-ABC, HLA-DR and ricinus communis aggulutinin lectin-1 (RCA-1), and actively phagocytosed latex beads . Gene expression and protein production of cytokines, chemokines and cytokine/chemokine receptors were investigated in the purified populations of human microglia . Normal unstimulated human microglia expressed constitutively mRNA transcripts for interleukin- 1beta (IL-1beta) -6, -8, -10, -12, -15, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and monocyte chemoattractant protein-1 (MCP-1), while treatment with lipopolysaccharide (LPS) or amyloid beta peptides (Abeta) led to increased expression of mRNA levels of IL-8, IL-10, IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and MCP-1 . Human microglia, in addition, expressed mRNA transcripts for IL-1RI, IL-1RII, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, and IL-15R . Enzyme-linked immunosorbent assays (ELISA) showed increased protein levels in culture media of IL-1beta, IL-8, TNF-alpha, and MIP-1alpha in human microglia following treatment with LPS or Abeta . Increased TNF-alpha release from human microglia following LPS treatment was completely inhibited with IL-10 pretreatment, but not with IL-6, IL-9, IL-12, IL-13, or transforming growth factor-beta (TGF-beta) . Present results should help in understanding the basic microglial biology, but also the pathophysiology of activated microglia in neurological diseases such as Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, stroke, and neurotrauma .

Bone, 2002 Jul, 31(1), 236 - 41
Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts; Reher P et al.; We have previously shown that the therapeutic range of ultrasound heals osteoradionecrotic bone and induces bone formation in vitro . It is well established that nitric oxide (NO) and prostaglandins are crucial early mediators in mechanically induced bone formation . The therapeutic range of ultrasound may act in the same way; therefore, we have investigated the effect of the therapeutic range of ultrasound on NO induction and prostaglandin E(2) (PGE(2)) production in vitro . Two ultrasound machines were evaluated, "traditional" (1 MHz, pulsed 1:4, tested at four intensities) and a "long-wave" (45 kHz, continuous, also tested at four intensities) devices . Ultrasound was applied to human mandibular osteoblasts for 5 min, and incubated at 37 degrees C for up to 24 h . The control group (sham insonated) was treated in the same way . NO was determined by measuring the nitrite concentration in the culture media colorimetrically, and PGE(2) was assayed by radioimmunoassay . Ultrasound produced a significant increase in both induced nitrite and PGE(2) production . The NO synthesis appeared to be via inducible NO synthase (iNOS) on the basis of the time course and levels of nitrite obtained, although the inhibition of other NOS isoforms by aminoguanidine cannot be excluded . PGE(2) synthesis appeared to be via COX-2 . With the 45 kHz machine, a significant increase in NO was achieved at three intensities, 5, 30, and 50 mW/cm(2) . The 1 MHz machine stimulated the synthesis of both NO and PGE(2), but was significant at only one dose (0.1 W/cm(2(SAPA))) . There was no difference between the two machines with regard to PGE(2) synthesis . The time-course experiment revealed peak production to be 12-18 h for both NO and PGE(2) . The therapeutic range of ultrasound stimulates both NO and PGE(2) synthesis by human osteoblasts, and the 45 kHz machine appeared to be more effective than the traditional short-wave length . These results may reflect the healing effect of ultrasound on fractures and osteoradionecrosis.

Pigment Cell Res, 2002 Aug, 15(4), 298 - 304
Morphological studies on microfilaments and their organizing center in killifish (Fundulus heteroclitus L.) melanophores; Kimler VA et al.; Fish chromatophores serve as excellent study models for cytoskeleton-dependent organelle translocations because the distribution of pigmentary organelles can be observed against a time frame by microscopy . In this study the distribution of microfilaments along with microtubules in cultured melanophores of the killifish (Fundulus heteroclitus Linneaus) are examined using whole-cell transmission electron microscopy (WCTEM), fluorescence, and laser scanning confocal microscopy . Dispersing, dispersed, aggregating and aggregated states of pigment are induced by adding either caffeine (for dispersion) or epinephrine (for aggregation) to the cells in a standard culture medium . The cells that exhibited a random melanosome distribution in the standard culture media without these two reagents, served as the control . The results indicate that: (i) a structure considered to be the actin-filament organizing center (AFOC) is in close proximity to the microtubule-organizing center (MTOC); (ii) the radial layout of microfilaments remains similar over four physiological states of pigmentary response with the exception of epinephrine-aggregated pigment, in which the aggregate blocks the viewing of the AFOC and central microfilament rays, yet radial microfilaments, whether central and/or peripheral, are apparent in all physiological states of distribution; and (iii) microfilaments serve, together with microtubules, as scaffolding for melanosomes which migrate in bi-directional rows on cross-bridges, thus shedding light on the mechanisms for orderly melanosome translocations in a structural continuum.

Clin Exp Immunol, 2002 Jul, 129(1), 86 - 91
Transmural pressure induces IL-6 secretion by intestinal epithelial cells; Kishikawa H et al.; Although intestinal epithelial cells (IECs) are known as an important source for IL-6, it is not known whether mechanical forces affect IL-6 production . We investigated how transmural pressure modulates IL-6 synthesis and activation of transcription factors in IECs . Pressure was loaded onto IEC-18 cells by introducing compressed helium gas into the cell culture flask for 1-48 h . IL-6 release into the culture media was determined by cell proliferation bioassay using an IL-6-dependent mouse hybridoma cell line (7TD1) . Exposure to pressure (80 mmHg) significantly enhanced IL-6 release into the culture media from IEC-18 cells at 12 h . Under control conditions, IL-6 secretion was directed to the basolateral side, but after exposure to pressure IL-6 secretion was increased in both the apical and basolateral sides . A nuclear factor kappa B (NF-kappaB) decoy reversed completely the pressure-induced increase of IL-6 secretion by IEC-18 cells . Pressure treatment enhanced IL-6 mRNA expression in IECs within 6 h . Pressure loading significantly enhanced the activation of both NF-kappaB and NF-IL-6 from 1h in the nuclear protein of IEC-18 cells as assessed by the electrophoretic mobility shift assay using FITC-conjugated specific primers . Increased phosphorylation of I-kappa B was also demonstrated in the cytosol of IEC cells within 1h by Western blot analysis . These results suggest a possible role for pressure loading in immune modulation of the intestinal mucosa by the stimulation of IL-6 release from intestinal epithelial cells.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(2), 121 - 125
Construction and Characterization of Recombinant Adenoviruses Expressing Biologically Active Human Brain-derived Neurotrophic Factor and Neurotrophin-3; Chen Q et al.; In order to deliver brain-derived neurotrophic factor(BDNF) and neurotrophin-3(NT3) into CNS to prevent or reduce degeneration of CNS neurons in human neurodegenerative diseases and neuron injuries, recombinant adenoviruses Ad-BDNF and Ad-NT3 were constructed . BDNF and NT3 genes were inserted into an E1-substituted adenovirus shuttle plasmid, respectively, and were driven by the human cytomegalovirus immediate-early gene promoter/enhancer . After cotransfection into 293 cells with the adenovirus plasmid pJM17, the recombinant adenovirus Ad-BDNF and Ad-NT3 were propagated in 293 cells via homologous recombination . After two rounds of CsCl centrifugation, the human recombinant adenovirus Ad-BDNF and Ad-NT3 were obtained with titer of 1x10(12) and 8x10(11) pfu/ml, respectively . To examine the expression of BDNF and NT3, HeLa cells were infected with Ad-BDNF and Ad-NT3, respectively . RT-PCR, Western blot and ELISA analysis results showed that BDNF and NT3 genes could be transcribed and translated in Ad-BDNF and Ad-NT3 infected HeLa cells . And the culture media containing 10% conditioned medium of Ad-BDNF and Ad-NT3 infected Hela cells could induce the neurite outgrowth from E8 dorsal root ganglion neurons.

J Nutr, 2002 Jul, 132(7), 1830 - 5
Selenium influences the turnover of selenocysteine tRNA({Ser}Sec) in Chinese hamster ovary cells; Jameson RR et al.; Selenocysteine transfer RNA (tRNA({Ser}Sec)) is a central molecule in the production of selenium-containing proteins, and may play a role in the regulation of their biosynthesis . Selenium concentration influences both the levels of tRNA({Ser}Sec) and the relative abundance of two isoforms . To study the mechanism by which selenium affects tRNA({Ser}Sec) levels, Chinese hamster ovary (CHO) cells were treated with the transcription inhibitor, actinomycin D, and tRNA({Ser}Sec) levels were determined by Northern blotting, primer extension and reverse-phase column chromatography . Turnover of tRNA({Ser}Sec) in CHO cells was faster than the total tRNA population . Supplementation of the culture media with selenium reduced turnover of tRNA({Ser}Sec), but did not influence turnover of a randomly selected serine tRNA . Inhibition of transcription with actinomycin D resulted in a relative increase in the abundance of the isoform containing methylcarboxymethyl-5'-uridine-2'-O-methylribose in the wobble position of the anticodon . Primer extension studies, which permitted the independent evaluation of the tRNA({Ser}Sec) arising from the introduced mouse gene and that derived from the host CHO gene, indicated an accelerated decline in tRNA({Ser}Sec) derived from both the transfected and the native gene . These results provide additional insight into the levels of regulation that control the translation of selenium containing proteins in mammalian cells.

Clin Microbiol Rev, 2002 Jul, 15(3), 401 - 13
In vitro cultivation of microsporidia of clinical importance; Visvesvara GS; Although attempts to develop methods for the in vitro cultivation of microsporidia began as early as 1937, the interest in the culture of these organisms was confined mostly to microsporidia that infect insects . The successful cultivation in 1969 of Encephalitozoon cuniculi, a microsporidium of mammalian origin, and the subsequent identification of these organisms as agents of human disease heightened interest in the cultivation of microsporidia . I describe the methodology as well as the cell lines, the culture media, and culture conditions used in the in vitro culture of microsporidia such as Brachiola (Nosema) algerae, Encephalitozoon cuniculi, E . hellem, E . intestinalis, Enterocytozoon bieneusi, Trachipleistophora hominis, and Vittaforma corneae that cause human disease.

Clin Microbiol Rev, 2002 Jul, 15(3), 374 - 89
Cultivation of clinically significant hemoflagellates; Schuster FL et al.; The hemoflagellates, Trypanosoma spp . and Leishmania spp., are causal agents of a number of parasitic diseases having a major impact on humans and domestic animals over vast areas of the globe . Among the diseases are some of the most pernicious and deadly of human afflictions: African sleeping sickness, Chagas' disease, kala-azar, and Oriental sore . The organisms have complex, pleomorphic life cycles typically involving a vertebrate and an invertebrate host, the latter serving as a vector . In the vertebrate host, they are primarily blood and tissue parasites . In their transition from one host to another, the hemoflagellates undergo morphological, physiological, and biochemical changes that facilitate their growth and subsequent transmission . A major goal in the study of the hemoflagellates has been the cultivation in vitro of both vertebrate and invertebrate stages of the organisms . The first types of media used in their cultivation, and still useful for establishment of cultures, were undefined and contained a complex of ingredients . These gave way to semidefined formulations which included tissue culture media as a base and, as a next step, addition of tissue culture cells as a feeder layer to promote parasite growth . More recently developed media are completely defined, having replaced the feeder cells with various supplements . Serum, a sometimes-variable component of the media, can be replaced by various serum substitutes . This review focuses on the hemoflagellates that infect humans, describing stages in the development of media leading to the fully defined formulations that are now available for the cultivation of many of these organisms.

Clin Microbiol Rev, 2002 Jul, 15(3), 355 - 64
Cultivation of plasmodium spp; Schuster FL; Cultivation of both human and non-human species of Plasmodium spp., the causal agent of malaria, has been a major research success, leading to a greater understanding of the parasite . Efforts at cultivating the organisms in vitro are complicated by the parasites' alternating between a human host and an arthropod vector, each having its own set of physiological, metabolic, and nutritional parameters . Life cycle stages of the four species that infect humans have been established in vitro . Of these four, P . falciparum remains the only species for which all stages have been cultured in vitro; different degrees of success have been achieved with the other human Plasmodium spp . The life cycle includes the exoerythrocytic stage (within liver cells), the erythrocytic stage (within erythrocytes or precursor reticulocytes), and the sporogonic stage (within the vector) . Culture media generally consist of a basic tissue culture medium (e.g., minimal essential medium or RPMI 1640) to which serum and erythrocytes are added . Most of the efforts have been directed toward the stage found in the erythrocyte . This stage has been cultivated in petri plates or other growth vessels in a candle jar to generate elevated CO(2) levels or in a more controlled CO(2) atmosphere . Later developments have employed continuous-flow systems to reduce the labor-intensive nature of medium changing . The exoerythrocytic and sporogonic life cycle stages have also been cultivated in vitro . A number of avian, rodent, and simian malarial parasites have also been established in vitro . Although cultivation is of great help in understanding the biology of Plasmodium, it does not lend itself to use for diagnostic purposes.

Cytokine, 2002 Apr 21, 18(2), 92 - 7
Glutamine decreases interleukin-8 and interleukin-6 but not nitric oxide and prostaglandins e(2) production by human gut in-vitro; Coeffier M et al.; BACKGROUND: Glutamine modulates cytokine production in various tissues but its effects on the production of other inflammatory mediators such as eicosanoids and nitric oxide have not been investigated in human gut . AIM: To evaluate the influence of glutamine on interleukin (IL)-8, IL-6, nitric oxide and prostaglandin E(2) production by human gut . METHODS: Ten fasted volunteers received either enteral glutamine or isonitrogenous amino acids over 6 h in a cross-over design . Series of duodenal biopsies were frozen or cultured for 24 h with 0.5 or 5 mM of glutamine or amino acids . IL-6, IL-8 and PGE(2) were measured in culture media by ELISA and nitrites by Griess assay . mRNA levels for IL-6, IL-8, Cyclooxygenase-2 and NO synthase-2 were assessed in biopsies by RT-PCR . Results in percent, (median {range}) were compared by Wilcoxon test . RESULTS: Glutamine decreased IL-8 and IL-6 in-vitro production: 63 {2-173} vs 100 {19-177} and 37 {5-489} vs 100 {33-431}, both P<0.05 . IL-8 mRNA level also decreased in biopsies cultured with 5 mM glutamine: 26 {13-142} vs 92 {34-215}, P<0.05 . Nitrites and PGE(2) concentrations were not significantly affected by glutamine . CONCLUSION: Glutamine has a specific inhibitory effect on pro-inflammatory cytokine production in the gut and may contribution to the modulation of intestinal inflammation .

Nutr Cancer, 2001, 41(1-2), 119 - 25
Green tea catechins and vitamin E inhibit angiogenesis of human microvascular endothelial cells through suppression of IL-8 production; Tang FY et al.; Epidemiological and animal studies have indicated that consumption of green tea and high vitamin E intake are associated with a reduced risk of developing certain forms of cancer . However, the inhibitory mechanism of green tea catechins and vitamin E in angiogenesis, an important process in tumor growth, has not been well established . In the present study, alpha-tocopherol and several major catechins of green tea (catechin, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate) were tested for their ability to inhibit tube formation in vitro using a model in which human microvascular endothelial cells were exposed to a constant rate of a physiologically low level of H2O2 . In this model, the production of interleukin (IL)-8 by human microvascular endothelial cells at a low level of H2O2 was required for angiogenesis, as assessed by tube formation in three-dimensional gel in culture . Vitamin E (d-alpha-tocopherol, 40 microM) in the culture media significantly reduced IL-8 production and angiogenesis . Among the green tea catechins, epigallocatechin (0.5-1 microM) was the most effective in reducing IL-8 production and inhibiting angiogenesis . These results suggest that consumption of green tea catechins or supplemental intake of vitamin E may have preventive effects on tumor development, mediated, at least in part, through inhibition of angiogenesis via suppression of IL-8 production.

Rev Med Chir Soc Med Nat Iasi, 2001 Jul-Sep, 105(3), 565 - 9
{Magnetic liquid influence upon some plant species of pharmaceutical interest}; Pavel A et al.; It was accomplished a study on the influence of a petroleum magnetic liquid upon two plant species of pharmaceutical interest: Papaver somniferum L . and Chelidonium majus L . Experimental observation aimed: callus accumulation, seed germination, mitotic index and fluorescence of the photosynthesis pigments . The plant samples were taken from in vitro cultures obtained from different explant types while the magnetic liquid was added in the culture media in low concentrations (ml/l) . The germination test showed a positive influence of the magnetic liquid, the cell division test revealed an increased mitotic index, callus accumulation rate is enhanced while the fluorescence spectra showed maxima shift for the samples in comparison to the controls.

Nucl Med Biol, 2002 Jul, 29(5), 537 - 45
In vitro and in vivo characteristics of a human colon cancer cell line, SNU-C5N, expressing sodium-iodide symporter; Min JJ et al.; Rat NIS (rNIS) genes were transfected into a human colon cancer cell line (SNU-C5) by lipofection . The transfected cells (SNU-C5N) exhibited an increase 125I uptake to a level 10 times higher than the untransfected SNU-C5 cells . The addition of 300 microM DIDS, an anion channel blocker, to the culture media led to a 2.35 times increase of 125I uptake in the cells . For the first 10 minutes, up to 70% of the cellular radioactivity was released into the medium . In the biodistribution study using SNU-C5N-xenografted mice, the %ID/g of the SNU-C5N tumors at 1, 3, 6, and 12 h after the 125I injection were 4.43%, 1.09%, 1.05%, and 0.05%, respectively, which were significantly higher than those for the SNU-C5 tumors (P<0.05) . In tumor imaging, the SNU-C5N-xenografted tumor was clearly visible . In this study, NIS lipofection is efficient for triggering significant iodide uptake by a nonthyroidal tumor . However, for an increased therapeutic effect, the key issue is iodide retention in the target tissue.

Biomed Sci Instrum, 2002, 38, 89 - 94
Release of inflammatory cytokines by macrophages and synovial cells challenged with tumor necrosis factor; Tucci MA et al.; Late aseptic loosening of total joint implants continues to be a common cause of implant failure . However, the pathophysiology of implant loosening remains controversial as to which factors at the implant tissue interface plays a crucial role in implant failure . The most prominent features of the foreign body membrane obtained from patients undergoing revision hip surgery were the presence of lymphocytes, histiocytes, giant cells, and immature collagen formation . The inflammatory sites are often characterized by the infiltration of activated lymphocytes and macrophages into the synovial membrane followed by proliferation of the synovial cells . The present study was conducted to determine if synovial cells in addition to macrophage cells are activated by tumor necrosis factor (TNF), which ultimately leads to foreign body membrane proliferation and ultimately joint space destruction . Macrophages or synovial cells were seeded at a density of 1 x 10(6) cells/ml and 5 ml were placed onto a 12 mm culture dish . The cells were challenged with either tissue culture media or media containing 2 ng/ml LPS or various doses of TNF (0.5, 5 and 50 ng/ml) . The result indicated that both cell types were able to produce the inflammatory cytokines (IL-1 and IL-8 as early as 8 hours after a challenge with peak production occurring between 18-24 hours) . The data suggest that both cell types are capable of eliciting inflammatory mediators, which can ultimately lead to joint destruction.

Gastrointest Endosc, 2002 Jul, 56(1), 72 - 7
Effects of adherence factors and human bile on bacterial attachment and biliary stent blockage: an in vitro study; Leung JW et al.; BACKGROUND: Bacterial attachment plays an important role in the initiation of biliary sludge formation and stent blockage . In vitro studies were conducted to determine the effects of adherence factors, namely pili and glycocalyx production, and culture media, including brain heart infusion broth, modified Vogel and Bonner medium, and human bile, on the adherence of Escherichia coli to plastic stents . METHODS: Clinical isolates of E coli with different adherence mechanisms, that is, piliated (P+) or nonpiliated (P-), glycocalyx producing (G+) and nonglycocalyx producing (G-), were obtained from clogged stents . Adherence studies were conducted by using the modified Robbins device, and stents were removed at regular intervals to determine the number of attached bacteria/cm(2) with the viable plate count method . Polyethylene stents were used to compare the adherence curves of E coli with different adherence factors in brain heart infusion broth . The effects of different culture media on the adherence of P+G+ E coli to polyethylene stents were determined . In addition, the adherence of P+G+ E coli to different plastics in brain heart infusion broth and human bile was compared . RESULTS: P+G+ E coli adhered better than P-G+ and P-G- E coli to polyethylene stents . Modified Vogel and Bonner medium, which stimulates glycocalyx production, enhanced the attachment of P+G+ E coli, whereas human bile decreased E coli attachment to polyethylene stents, despite an increase in glycocalyx production . There was a difference in adherence of P+G+ E coli to polyethylene, polyurethane, and Teflon stents in brain heart infusion broth, but the differences were nullified in the presence of human bile . CONCLUSIONS: P+G+ E coli with both adherence factors adhere best to plastic stents . Media such as modified Vogel and Bonner medium that stimulate glycocalyx production also enhance bacterial attachment . The toxic effects of bile salts in human bile on the bacteria might alter the adherence mechanism and reduce E coli attachment.

J Pathol, 2002 May, 197(1), 72 - 81
Expression and regulation of collagenase-2 (MMP-8) in head and neck squamous cell carcinomas; Moilanen M et al.; MMP-8 (collagenase-2) is the most effective collagenase to initiate type I collagen degradation . Since initiation of lysis of the surrounding collagen matrix is an essential prerequisite for carcinoma cells to spread, this study investigated the expression of MMP-8 in squamous cell carcinoma (SCC) of the head and neck in vivo and in vitro . Most of the recently established head and neck carcinoma cell lines (22/25), corresponding tumour (5/7) and dermal (2/2) fibroblasts, commercial tongue carcinoma (HSC-3 and SCC-25), and transformed keratinocyte cell lines of the tongue (IHGK) and skin (HaCaT) expressed MMP-8 mRNA analysed by the PCR method . Western blotting revealed a latent 50 kD band in concentrated culture media of carcinoma cells and corresponding tumour and dermal fibroblasts . The expression of immunoreactive MMP-8 protein was reduced 30% by transforming growth factor beta-1 (TGF-beta1) at 1 ng/ml concentration and 60% at 10 ng/ml concentration, but up-regulated 2- and 2.5-fold after 10 nM and 100 nM phorbol 12-myristate 13 acetate (PMA), respectively . Immunohistological staining localized MMP-8 protein in a few malignant invading tumour cell islands, certain fibroblasts, polymorphonuclear neutrophils (PMNs), and plasma cells . In situ hybridization revealed a faint sporadic signal in carcinoma cells of all eight tissue sections analysed . It is concluded that tissue from head and neck carcinomas can express MMP-8 both in vivo and in vitro . Since the amount of MMP-8 in carcinoma and stromal cells is rather low, MMP-8 may have a potential role, with other collagenases, in the proteolysis of connective tissue associated with the spreading of invasive carcinoma.

J Ind Microbiol Biotechnol, 2002 Jul, 29(1), 6 - 9
In vitro colonization of hydrophilic contact lenses by Aspergillus niger; Marques-Calvo MS; In vitro colonization of hydrophilic contact lenses by Aspergillus niger was investigated . Five strains of the fungus, four polymers, two culture media and four incubation periods were considered for analysis . Only the 2700 strain colonized the lenses . The degrees of adhesion and invasion varied significantly according to the characteristics of the culture under investigation.

Biol Reprod, 2002 Jul, 67(1), 88 - 98
Potassium channel antagonists influence porcine granulosa cell proliferation, differentiation, and apoptosis; Manikkam M et al.; This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells . The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX) . Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively . Granulosa cell apoptosis was evaluated using flow cytometry . Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase . The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells . However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis . All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide . In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell . These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis . Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

J Anim Sci, 2002 Jun, 80(6), 1616 - 22
Technical note: epimerization of ergopeptine alkaloids in organic and aqueous solvents; Smith DJ et al.; Purified ergopeptine alkaloids are often used in studies related to tall fescue toxicosis without regard to epimerization that occurs when ergopeptines are solvated . The objectives of this study were to measure the rates of alpha-ergocryptine epimerization to alpha-ergocryptinine at room temperature and at -40 degrees C, and to measure the rate of ergovaline epimerization to ergovalinine at 37 degrees C . Alpha-ergocryptine tartrate was stable (< 0.5% epimerization) in protic or aprotic solvents when stored at -40 degrees C for 20 to 52 d . At room temperature, alpha-ergocryptine epimerization in chloroform did not occur; epimerization was modest in acetone and acetonitrile (< 5%) but was substantial in methanol (78% by 38 d) and in a 70:30 water methanol mix (47% by 42 d) . Ergovaline epimerization to ergovalinine occurred at 37 degrees C in 0.1 M phosphate buffers (pH 3, 7.5, and 9) in 9% aqueous solutions of fetal bovine serum (FBS), and in water, methanol, and acetonitrile . The degree of epimerization at 37 degrees C was solvent-dependent . Epimerization rates with respect to time were roughly linear in phosphate buffer (pH 3 only), water, methanol, and acetonitrile; epimerization rates resembled first-order kinetics in phosphate buffers (pH 7.5 and 9) and in the presence of FBS (pH 3, 7.5 and in Dulbecco's culture media) . Epimerization equilibria (48 to 63% ergovaline) were reached within approximately 1 to 19 h . Results from this study indicate that researchers conducting studies with purified ergopeptines should carefully control the storage conditions of solvated ergopeptines and measure isomeric composition under the actual experimental conditions used in experiments.

J Pediatr Surg, 2002 Jul, 37(7), 1051 - 7; discussion 1051-7
Adeno-associated virus (AAV)-mediated gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture; Lim FY et al.; BACKGROUND/PURPOSE: Since the discovery of the cystic fibrosis transmembrane regulator (CFTR) gene, cystic fibrosis has been an attractive target for gene therapy . Postnatal gene transfer in the respiratory epithelium has been difficult and particularly inefficient in the submucosal gland cells, the target cells for CFTR gene transfer . The authors hypothesized that during development, there is a favorable environment for fetal gene therapy with fewer physical barriers to efficient gene transfer and more accessible epithelial and submucosal gland precursor cells . The authors tested this hypothesis in a novel human fetal tracheal organ culture system using a serotype 2 recombinant AAV that contains an enhanced green fluorescent protein (eGFP) reporter gene (AAV-CMV-eGFP) . METHODS: Human fetal tracheas at between 16 and 20 weeks' gestation age were used in this study . The distal end of each trachea was ligated and secured in an upright position with the open proximal end facing up . Only the ante-lumenal surface was exposed to culture media . 5 x 10(9) particles of AAV-CMV-eGFP were administered intratracheally through the open end . Fetal tracheas were maintained in tracheal organ culture media and harvested at either 2 weeks (n = 3) or 4 weeks (n = 7) after injection . The fetal tracheas were processed for routine H&E, standard electron microscopy (EM), and fluorescence microscopy for analysis of eGFP transgene expression . RESULTS: Histology confirmed the preservation of structural integrity out to 4 weeks of fetal tracheal organ culture . EM showed intact tight junctions of the apical respiratory epithelium . At 2 weeks after AAV-CMV-eGFP injection, there was minimal transgene expression . However, at 4 weeks there was extensive transgene expression in not only the respiratory surface epithelium but also the submucosal gland cells of the human fetal tracheal organ culture . Transgene expression was seen in nearly all cells in the submucosal glands . CONCLUSIONS: AAV-mediated gene transfer in human fetal tracheal organ culture was highly efficient with excellent transgene expression at 4 weeks in both respiratory surface epithelium and submucosal gland cells . This highly efficient gene transfer may result from fewer physical barriers and more accessible target precursor cells . These results are consistent with more efficient gene transfer in fetal tracheobronchial epithelium and show the potential for fetal gene therapy using AAV for the treatment of congenital airway disease such as cystic fibrosis .

J Pineal Res, 2002 Apr, 32(3), 135 - 42
The neuroprotective activities of melatonin against the Alzheimer beta-protein are not mediated by melatonin membrane receptors; Pappolla MA et al.; Exposure of neuronal cells to the Alzheimer's amyloid beta protein (Abeta) results in extensive oxidative damage of bio-molecules that are profoundly harmful to neuronal homeostasis . It has been demonstrated that melatonin protects neurons against Abeta-mediated neurotoxicity, including cell death and a spectrum of oxidative lesions . We undertook the current study to determine whether melatonin membrane receptors are involved in the mechanism of neuroprotection against Abeta neurotoxicity . For this purpose, we characterized the free-radical scavenging potency of several compounds exhibiting various affinities for melatonin membrane receptors (MLT 1a and 1b) . Abeta-mediated neurotoxicity was assessed in human neuroblastoma cells and in primary hippocampal neurons . In sharp contrast with melatonin, no neuroprotection against Abeta toxicity was observed when we used melatonin membrane receptor agonists that were devoid of antioxidant activity . In contrast, the cells were fully protected in parallel control experiments when either melatonin, or the structurally unrelated free-radical scavenger phenyl-N-t-butyl nitrone (PBN), were added to Abeta-containing culture media . This study demonstrates that the neuroprotective properties of melatonin against Abeta-mediated toxicity does not require binding of melatonin to a membrane receptor and is likely the result of the antioxidant and antiamyloidogenic features of the agent.

Free Radic Res, 2002 Mar, 36(3), 285 - 94
Roles of bioavailable iron and calcium in coal dust-induced oxidative stress: possible implications in coal workers' lung disease; Zhang Q et al.; Marked regional differences in prevalence of pneumoconiosis are apparent in the US despite comparable dust exposure . In the present study, we examined the ability of 28 coal samples to release bioavailable iron (BAI) and calcium, as well as other metals such as Cr, Ni, Cu, and Co, from three coal mine regions in Utah (UT), West Virginia (WV), and Pennsylvania (PA), respectively . BAI is defined as iron (both Fe2+ and Fe3+) released by the coals in 10 mM phosphate solution, pH 4.5, which mimics conditions of the phagolysosomes in cells . We found that coals from the UT, WV, and PA regions released average levels of BAI of 9.6, 4658.8, and 12149 parts per million (ppm, w/w), respectively, which correlated well with the prevalence of pneumoconiosis from that region (correlation coefficient r = 0.92) . The low levels of BAI in the UT coals were due to the presence of calcite (CaCO3), which was shown to be preferentially acid solubilized before iron compounds . Release of iron by two coal samples from the PA and UT regions was further examined in vitro in human lung epithelial A549 cells . We found that the coal from PA, with a high prevalence of pneumoconiosis, released BAI in a dose-dependent manner, both in tissue culture media and in A549 cells . At 2 microg/cm2, levels of lipid peroxidation induced by the PA coal were increased 112% over control cells at 24 h treatment, and were sustained at this level for 3 days . The coal from UT, with a low prevalence of pneumoconiosis, induced a marginal increase in cellular iron at 5 and 10 microg/cm2 treatments and had no effect on lipid peroxidation . Calcium levels in the cells treated with the PA and UT coals were 8.6 and 11.5 micromoles/10(6) cells, respectively, and were significantly higher than that in the controls (5.3 micromoles/10(6) cells) {corrected} . Our results suggest that the differences in the BAI content in the coals may be responsible for the observed regional differences in the prevalence of pneumoconiosis . Therefore, BAI may be a useful characteristic of coal for predicting coal's toxicity.

Biomaterials, 2002 Jul, 23(14), 2989 - 96
Colonization of ion-modified polyethylene with vascular smooth muscle cells in vitro; Walachova K et al.; Polyethylene (PE) foils were implanted with 40 and 150 keV Ar+ ions to the fluences from 1 x 10(13) to 1 x 10(15) cm(-2) . Production of conjugated double bonds, characterizing degradation of the PE surface layer, was studied using UV-VIS spectroscopy . Wettability of the PE surface, determined by conventional goniometric techniques, was shown to be an increasing function of both ion energy and fluence . It was also increased after exposure of PE to serum-supplemented cell culture media . Cell culture experiments showed that the ion irradiation significantly increased the adherence of vascular smooth muscle cells (VSMC) and their subsequent growth on the PE surface . On day 1 after seeding, the number of initially adhered VSMC exhibited two maxima . On day 3 after seeding . these maxima disappeared, which was partially due to a significantly shorter doubling time of VSMC . On the other ion-modified samples . the doubling time did not differ significantly from that on the unmodified PE . Enzyme-linked immunosorbent assay revealed increased concentration of talin, a protein of focal adhesion plaques, and alpha-actin, a marker of VSMC differentiation, in cells on ion-implanted surfaces . It can be concluded that the ion irradiation supports the adhesion and differentiation of VSMC without excessive proliferation of these cells.

Prikl Biokhim Mikrobiol, 2002 May-Jun, 38(3), 243 - 7
{Dependence of activities of polysaccharide hydrolases and oxidases from Cerrena unicolor on the source of carbon and aromatic acids in culture media}; Elisashvili V et al.; The activities of carboxymethylcellulase and xylanase in the higher basidial fungus Cerrena unicolor grown in avicel-containing medium reached 1.95 and 1.50 units per mg protein, respectively, whereas in mannitol-containing medium they ranged from 0.02 to 0.05 units per mg protein . The activity of fungal beta-glucosidase depended on the carbon source in the culture medium and ranged from 2.1 units per mg protein in the presence of mannitol to 17.3 units per mg protein in the presence of avicel . In contrast to polysaccharides, easily metabolizable substrates (cellobiose, mannitol, and glucose) provided the highest rates of secretion of laccase (52.7-123.5 ncat per mg protein) and ligninase (22-106 units per mg protein) . The addition of tangerine pomace, a substrate enriched with aromatic compounds, to the culture medium caused an increase in the rate of bio-synthesis of laccase and ligninase to 862 ncat per ml and 557 units per ml, respectively . Aromatic compounds such as p-xylidine and veratric aldehyde increased the laccase activity of C . unicolor IBB 62 from 7.9 to 23.6 and 18.3 ncat per mg protein, respectively . Veratryl alcohol caused a sevenfold increase in the activity of Mn-dependent peroxidase in the culture medium.

J Neurochem, 2002 Jun, 81(6), 1166 - 75
Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y; Brakch N et al.; To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells . The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence . Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase . The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways . ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent . The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal.

J Neurochem, 2002 Jun, 81(6), 1141 - 51
Alteration of amino acid metabolism in neuronal aggregate cultures exposed to hypoglycaemic conditions; Honegger P et al.; The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones . Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult . Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency . Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased . Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage . Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage . Both lactate and glutamine were readily oxidized in these neuronal cultures . The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions . This results in increased ammonia production and neuronal damage.

J Androl, 2002 Jul-Aug, 23(4), 522 - 8
Measurement of volume changes in mouse spermatozoa using an electronic sizing analyzer and a flow cytometer: validation and application to an infertile mouse model; Yeung CH et al.; The importance of sperm volume has recently been highlighted in a knockout mouse model in which infertility was caused by defects in volume reguiation, which led to sperm transport failure in the female tract . Inhibition of volume regulation by human sperm, resulting in failure of penetration of cervical mucus in vitro, has also been reported . The present work aims to establish a sensitive and convenient method for monitoring changes in sperm volume for functional studies . Mature murine sperm obtained from the cauda epididymidis were analyzed by flow cytometry for their forward and side (90 degrees C) scatter of a 488-nm excitation wavelength laser, and the data were compared with volumes measured by electronic sizing using a Coulter counter . Changes in cell volume were induced by releasing or diluting sperm into culture media of various osmolalities (208-520 mmol/kg) . Forward scatter signal (FSS) intensity correlated well with volume measurement obtained by a Coulter counter (R =.83; P <.001), confirming that FSS reflects Coulter counter findings as for somatic cells . Sperm swelling was also induced by the presence of quinine, a wide-spectrum channel blocker, in a medium of 330 mmol/kg, which is similar to the osmolality of uterine fluid . The effect of quinine on sperm volume was more obvious when analyzed by flow cytometry than by electronic sizing . This effect was even more marked after dead sperm identified by fluorescent dye were eliminated from analysis using flow cytometry . Swelling was characterized by an increase in forward scatter and side scatter, generating a subpopulation of sperm that correlated well (R =.79; P <.0001) with the population of sperm exhibiting an angulation of the tail, which is a morphological manifestation of swollen murine sperm . Flow cytometric analysis revealed that infertile sperm released from the cauda epididymidis of c-ros knockout mice were significantly larger than those of fertile sperm from heterozygous mice . This finding directly substantiates the suggestion that infertile sperm are defective in their volume regulation . Laser scatter analysis of viable murine sperm by flow cytometry offers a convenient and sensitive method for the study of sperm volume regulation.

J Reprod Immunol, 2002 May-Jun, 55(1-2), 85 - 100
Physiology and culture of the human blastocyst; Gardner DK et al.; The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage . Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization . Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period . Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus . Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred . With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 451 - 5
Osteoprotegerin secretion from prostate cancer is stimulated by cytokines, in vitro; Penno H et al.; Osteoprotegerin (OPG), a member of the tumor necrosis receptor family, is produced by various tissues and inhibits osteoclast differentiation and activity . Since the metastasis of prostate cancer to bone often induces osteosclerosis, the possibility that these tumor cells secrete OPG is of interest . We have investigated whether the prostate cancer cell lines LNCaP, PC-3, and DU-145 produce and secrete OPG in vitro and if the production might be regulated by cytokines involved in remodeling of bone . OPG transcripts were detected by RT-PCR in all cell lines . OPG in culture media was analyzed by ELISA . In all three lineages, treatment with tumor necrosis factor-alpha and interleukin-1 beta dose dependently (5-5000 pM) stimulated the OPG secretion . Treatment with tumor necrosis factor-beta in increasing concentrations (1-1000 pM) stimulated OPG secretion in PC-3 but had no effect on the DU-145 and LNCaP cells . Dexamethasone (100 pM) had a small, but not significant, inhibitory effect on OPG secretion from DU-145 and LNCaP . In human non-malignant prostate cells, used as controls, there was no effect of IL-1 or TNFs on the secretion rate of OPG.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 55 - 60
The amino-terminal region of insulin-like growth factor binding protein-3, (1-95)IGFBP-3, induces apoptosis of MCF-7 breast carcinoma cells; Bernard L et al.; In an earlier study, we reported that an N-terminal proteolytic fragment ((1-95)IGFBP-3) corresponding to the first 95 residues of human insulin-like growth factor binding protein-3 (IGFBP-3) inhibits proliferation in a variety of fibroblasts . With a view to investigating its cytostatic capacity in carcinoma cells, we transiently transfected MCF-7 breast adenocarcinoma cells with an expression vector containing (1-95)IGFBP-3 cDNA . The transfected cells secreted a hyper-glycosylated form of (1-95)IGFBP-3 . Twenty-four hours after transfection, cell morphology and viability were similar in control and (1-95)IGFBP-3-secreting cells . However, after 48 h, (1-95)IGFBP-3-secreting cells were apoptotic, with marked cytoplasmic vacuolation and increased free histones in the cytoplasm . Culture media conditioned by (1-95)IGFBP-3-secreting cells also induced morphological changes and apoptosis in wild-type MCF-7 cells, indicating that (1-95)IGFBP-3 was responsible for the effects observed . These results provide further evidence that the N-terminal proteolytic fragment of IGFBP-3 has a functional role.

Biochem Biophys Res Commun, 2002 May 17, 293(4), 1183 - 90
Formation of MUC1 metabolic complex is conserved in tumor-derived and normal epithelial cells; Julian J et al.; MUC1 is abundantly expressed at the surface of epithelial cells in many tissues and their carcinomas . In humans, genetic polymorphism and differential splicing produce isoforms that may contribute to MUC1 participation in protection of the cell surface, modulation of cell-cell interactions, signaling, and metastasis . Biosynthetic and processing studies in tumor-derived cell lines indicate that cell surface MUC1 consists of a non-covalently associated heterodimer of separate cytoplasmic tail and extracellular domains . This heterodimer results from a single precursor proteolytically cleaved intracellularly . To determine whether similar processing of this isoform occurs in normal epithelial cells, we have examined cell-associated MUC1 and MUC1 released into medium by normal human uterine, mammary, and prostate epithelial cells . Cell-associated MUC1/REP was extracted as an SDS labile complex which was resistant to dissociation by boiling, urea, sulfhydryl reduction, peroxide, high salt, or low pH and was present in all normal cells examined . Addition of various proteolytic inhibitors during extraction had no effect on the proportion of this complex detected . MUC1 released into the medium was not associated with a particulate fraction (100,000g insoluble) and lacked the cytoplasmic tail . MUC1/REP and the MUC1 isoform lacking the transmembrane/cytoplasmic tail region, MUC1/SEC, mRNA were detected in all normal cells examined indicating that both shed and secreted MUC1 are likely to contribute to soluble forms found in culture media . (c) 2002 Elsevier Science (USA).

Reproduction, 2002 Jun, 123(6), 891 - 8
Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in cultured rat ovarian follicles; Duggal PS et al.; Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction . It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake . However, feed restriction alone does not inhibit ovulation . Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation . In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated . The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro . In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined . A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma . The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake . Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation . In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)) . The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant) . DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media . These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production . In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

Biotechnol Prog, 2002 May-Jun, 18(3), 476 - 82
Evaluation of infection parameters in the production of replication-defective HSV-1 viral vectors; Ozuer A et al.; Herpes simplex virus type-1 (HSV-1) is a neurotrophic human pathogen that establishes life-long latency in the nervous system . Our laboratory has extensively engineered this virus to retain the ability to persist in neurons without expression of lytic genes or disease phenotype . Highly defective, replication-incompetent HSV mutants are thus potentially ideal for transfer of therapeutic transgenes to human nerves where long-term therapy of nervous system disease may be provided . A prerequisite for using recombinant HSV vectors for therapeutic gene delivery to humans is the development of methods for large-scale manufacture of HSV vectors . Here we report studies to identify infection parameters that result in high-yield production of immediate early gene deletion mutant HSV vectors in complementing cells that supply the deleted essential viral functions in trans . Virus yield was correlated with various culture media conditions that included pH, glucose metabolism, and serum levels . The results demonstrated that systematic media exchange to remove lactate derived from high-level glucose consumption, maintenance of tissue culture pH at 6.8, and the use of 5% fetal bovine serum gave the highest yield of infectious virus . The data indicate that these are important parameters to consider for high-yield, large-scale virus production.

Atherosclerosis, 2002 Jul, 163(1), 59 - 68
Simvastatin, an HMG-CoA reductase inhibitor, induces the synthesis and secretion of apolipoprotein AI in HepG2 cells and primary hamster hepatocytes; Bonn V et al.; Clinical studies have recently suggested that statin treatment may beneficially elevate plasma concentrations of high density lipoprotein (HDL)-cholesterol in patients with hyperlipidemia . Here, we have investigated the effect of a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase on the synthesis and secretion of apolipoprotein AI (apoAI) in two model systems, HepG2 cells and primary hamster hepatocytes . Cultured cells were incubated with different doses of simvastatin (0.1-10 microM) for a period of 18 h . A dose-dependent increase in synthesis and secretion of apoAI was observed in both cell types . There was a significant increase in the synthesis of apoAI in HepG2 cells (44.3+/-12.1%), and hamster hepatocytes (212+/-2%) after treatment with 10 microM of the statin . The increase in apoAI synthesis appeared to result in a higher level of apoAI secreted into the culture media in both cell types (49.2+/-7.8% in HepG2, 197+/-0.2% in hamster hepatocytes) . ApoAI mRNA levels were also significantly increased in both cell types in response to statin treatment . Control experiments with transferrin confirmed specificity of the effect on apoAI secretion . Analysis of a density fraction containing HDL particles in culture media revealed an increase in HDL-associated apoAI of 94.3+/-2.1% in HepG2 cells and 27.0+/-0.03% in hamster hepatocytes following 10 microM simvastatin-treatment . Comparative studies of simvastatin and lovastatin indicated a differential ability to induce apoAI synthesis and secretion, with simvastatin having a more significant effect . Thus, acute statin treatment of cultured hepatocytes (transformed as well as primary) resulted in a significant upregulation of apoAI mRNA and apoAI synthesis, causing oversecretion of apoAI and HDL extracellularly . The stimulatory effect on apoAI synthesis and secretion may thus explain the clinical observation of an elevated plasma HDL-cholesterol level in hyperlipidemic patients treated with certain statins.

Water Res, 2002 Apr, 36(7), 1699 - 706
Biotransformation of 2,4,6-trinitrotoluene in a continuous-flow Anabaena sp . system; Pavlostathis SG et al.; Reductive transformation of 2,4,6-trinitrotoluene (TNT) was observed in a continuous-flow system of Anabaena sp . operated for 33 d with a 5.7 d hydraulic retention time and a range of influent TNT concentrations of 1-58 mg/l . The TNT removal efficiency of the continuous-flow system at the highest influent TNT concentration of 58 mg/l was 96.7 +/- 1.7% (mean +/- 95% confidence interval) . Culture chlorosis and growth inhibition were not observed during this study . The pseudo-first order TNT transformation rate constant values corresponding to the system performance range (0.14-0.46/h) were lower than the values previously recorded for batch Anabaena sp . cultures with less than 10 mg/l initial TNT concentrations, possibly due to an inhibition of the TNT transformation process by either TNT and/or TNT transformation products . Heterotrophic bacterial populations developed in the continuous-flow Anabaena sp . cultures also transformed TNT, but at a much lower rate than the Anabaena sp . Less than 1% of the overall TNT transformation observed in the continuous-flow system was attributed to the heterotrophic bacterial populations . The only TNT reduction products identified in both the culture media and in biomass extracts were azoxytetranitrotoluene isomers and low levels of aminodinitrotoluene isomers . TNT and TNT transformation products identified in the culture effluent and the biomass extract accounted for only about 24% of the TNT added to the system (on a molar basis) . Production of soluble, polar metabolites, uptake, partial mineralization and/or sequestration of TNT and its transformation products by Anabaena may be responsible for the relatively low contaminant recovery and mass balance observed in this study.

J Vasc Surg, 2002 Jun, 35(6), 1253 - 9
Rat and human aortic smooth muscle cells display differing migration and matrix metalloproteinase activities in response to dexamethasone; Pross C et al.; OBJECTIVE: The steroid dexamethasone inhibits neointimal hyperplasia development in rats but not in humans . This study investigates the differential effects of dexamethasone on rat and human smooth muscle cell migration and matrix metalloproteinase (MMP) activity . METHODS: Rat aortic smooth muscle cells were harvested from Sprague-Dawley rats . Human aortic smooth muscle cells were obtained from Clonetics . Boyden chamber migration assays were performed with chemoattractant (platelet-derived growth factor) and varying concentrations of dexamethasone (10(-9) to 10(-5) mol/L) . Zymography of culture media was used to assess MMP activity, and Western blot analysis was used for quantification of MMP-2 and tissue inhibitor of MMP-2 (TIMP-2) secretion . RESULTS: Dexamethasone inhibits rat aortic smooth muscle cell migration in a dose-dependent fashion . An increase in concentrations of dexamethasone does not effect human aortic smooth muscle cell migration . Rat aortic smooth muscle cell MMP-2 activity is inhibited with dexamethasone in a dose-dependent fashion, and human aortic smooth muscle cell MMP-2 activity is unchanged with dexamethasone . MMP-2 secretion is inhibited with dexamethasone in rat aortic smooth muscle cells but remains unaltered in human aortic smooth muscle cells . Dexamethasone increases rat aortic smooth muscle cell TIMP-2 secretion, and human aortic smooth muscle cell TIMP-2 secretion remains constant . CONCLUSION: Dexamethasone inhibits rat aortic smooth muscle cell migration, MMP-2 activity, and MMP-2 secretion and increases TIMP-2 secretion . These effects are not observed in human aortic smooth muscle cells . These findings may explain why dexamethasone inhibits neointimal hyperplasia in animal models but is ineffective in humans . Inhibition of human smooth muscle cell migration in vitro may be useful in predicting the effectiveness of future therapeutic agents for treatment of neointimal hyperplasia in humans.

J Biol Chem, 2002 Aug 16, 277(33), 29919 - 26 Epub 2002 May 31.
Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice; Gong JS et al.; We have reported previously (Michikawa, M., Fan, Q.-W., Isobe, I., and Yanagisawa, K . (2000) J . Neurochem . 74, 1008-1016) that exogenously added recombinant human apolipoprotein E (apoE) promotes cholesterol release in an isoform-dependent manner . However, the molecular mechanism underlying this isoform-dependent promotion of cholesterol release remains undetermined . In this study, we demonstrate that the cholesterol release is mediated by endogenously synthesized and secreted apoE isoforms and clarify the mechanism underlying this apoE isoform-dependent cholesterol release using cultured astrocytes prepared from human apoE3 and apoE4 knock-in mice . Cholesterol and phospholipids were released into the culture media, resulting in the generation of two types of high density lipoprotein (HDL)-like particles; one was associated with apoE and the other with apoJ . The amount of cholesterol released into the culture media from the apoE3-expressing astrocytes was approximately 2.5-fold greater than that from apoE4-expressing astrocytes . In contrast, the amount of apoE3 released in association with the HDL-like particles was similar to that of apoE4, and the sizes of the HDL-like particles released from apoE3- and apoE4-expressing astrocytes were similar . The molar ratios of cholesterol to apoE in the HDL fraction of the culture media of apoE3- and apoE4-expressing astrocytes were 250 +/- 6.0 and 119 +/- 5.1, respectively . These data indicate that apoE3 has an ability to generate similarly sized lipid particles with less number of apoE molecules than apoE4, suggesting that apoE3-expressing astrocytes can supply more cholesterol to neurons than apoE4-expressing astrocytes . These findings provide a new insight into the issue concerning the putative alteration of apoE-related cholesterol metabolism in Alzheimer's disease.

Theriogenology, 2002 Feb, 57(3), 1151 - 9
Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes; Nandi S et al.; Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes . Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse . Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish . The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h . The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope . Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium . Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days . Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture . These events mainly exhibited during 22-24 h of culture . Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.

Theriogenology, 2002 Apr 15, 57(7), 1839 - 54
In vitro culture of buffalo (Bubalus bubalis) preantral follicles; Gupta PS et al.; Growth of buffalo preantral follicles in culture was studied to investigate the effect of size of preantral follicles, individual or group culture, long-term culture of preantral follicles for (40 days), addition of human follicle stimulating hormone (FSH), insulin-transferrin-selenium (ITS), growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF), vaso active intestinal polypeptide (VIP) in culture media, and substitution of pregnant mare serum gonadotrophin (PMSG) for FSH as gonadotrophin source in culture media . Preantral follicles were isolated mechanically from ovaries of matured, nonpregnant slaughtered buffaloes and cultured in droplets of culture media under mineral oil in a 35 mm petri dish in a CO2 incubator (38-39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 15 days . Preantral follicle isolation and washing medium consisted of Minimum Essential Medium (MEM) supplemented with steer serum (10%), glutamine (2 mM), sodium pyruvate (0.23 mM), hypoxanthine (2 mM) and gentamycin (50 microg/ml), respectively . In Experiment 1, we placed isolated preantral follicles individually or in groups of 2-4 preantral follicles in 30 or 50 microl droplets, respectively, using two culture media: washing media and washing media + ITS (1%) + FSH (0.05 IU/ml), respectively . In Experiment 2, we grouped isolated preantral follicles were grouped into six different size classes: < or = 36, 37-54, 55-72, 73-90, 90-108 and > or = 109 microm . We cultured groups of 2-4 preantral follicles in washing media + ITS (1A) + FSH (0.05 IU/ml) in a CO2 incubator for 15 days . In Experiment 3, we allocated groups of 2-4 preantral follicles to 10 treatments: (1) only washing media, (2) washing media + FSH (0.05 IU/ml), (3) washing media + ITS (17%), (4) washing media + ITS (1%) + FSH (50 IU/ml), (5) washing media + ITS (1%) + EGF (50 ng/ml), (6) washing media + ITS (1%) + FSH (0.05 IU/ml) + EGF (50 ng/ml), (7) washing media + ITS (1%) + FGF (50 ng/ml), (8) washing media + ITS (1%) + FSH (0.05 IU/ml) + FGF (50 ng/ml), (9) washing media + ITS (1%) + VIP (50 ng/ml), and (10) washing media + ITS (1%) + FSH (0.05 IU/ml) + VIP (50 ng/ml) . In Experiment 4, based on the results of Experiment 3, we incubated preantral follicles from those treatments showing significantly (P < 0.05) higher growth up to 40 days . In Experiment 5, we allocated groups of 2-4 preantral follicles to two treatments: (1) washing media + PMSG (50 IU/ml), and (2) washing media + ITS (1%) + PMSG (50 IU/ml) and cultured in a CO2 incubator for 15 days . The results indicated that the preantral follicles cultured in groups had a higher growth rate (P < 0.05) than those cultured as individuals . ITS, FSH, PMSG and growth factors significantly (P < 0.05) promoted the growth of the preantral follicles . Following 40 days of culture, follicular architecture was preserved in nearly 17% of the follicles though there was no antrum formation . The growth rate of preantral follicles was lower in buffalo than in cattle.

Theriogenology, 2002 Apr 15, 57(7), 1765 - 79
Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes; Bogh IB et al.; In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial . The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes . During estrus, 19 pony mares were treated with 25 mg CEG . After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3) . Cumulus expansion rate was significantly affected by culture medium . The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%) . For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM) . An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3 . The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.

Southeast Asian J Trop Med Public Health, 2001, 32 Suppl 2, 31 - 5
In vitro development of Haplorchis taichui (Trematoda: Heterophyidae); Chaithong U et al.; Newly excysted metacercariae of Haplorchis taichui were cultured in a candle jar set at 37 degrees C . Both monophasic culture media {0.85% NaCl, RPMI 1640, RPMI 1640+10% fetal calf serum (FCS)} and diphasic culture media {RPMI 1640 + egg yolk agar, RPMI 1640 + 5%, 10% or 15% blood in blood agar (BA), RMPI 1640 + 5%, 10% and 15% FCS with 5% blood in BA} were used in vitro . Parasites survived for only 1 day in 0.85% NaCl without any development . In RPMI 1640 with egg yolk agar and RMPI 1640 + 5%, 10% FCS, the parasite survived for 3-5 days . In contrast, worms survived for 12-14 days in RPMI 1640 with blood agar without any change in result in a different concentration of blood in BA . The ovary and testes were observed after 3 days incubation in this media . Nevertheless, only 1 parasite in RPMI 1640 with 15% blood in BA had vitellaria and eggs at day 6 . RPMI 1640 with blood agar can be used as short-term maintenance for the in vitro culture of H . taichui . However, further studies are needed.

FASEB J, 2002 Jul, 16(9), 1102 - 4 Epub 2002 May 08.
Vitamin C matters: increased oxidative stress in cultured human aortic endothelial cells without supplemental ascorbic acid; Smith AR et al.; Because standard culture media for human aortic endothelial cells (HAEC) do not contain vitamin C, we hypothesized that HAEC may be under significant oxidative insult compared with the situation in vivo . To assess parameters of oxidative stress, intracellular vitamin C, glutathione (GSH), GSH/GSSG, and NAD(P)H/NAD(P)+ ratios, as well as oxidant appearance and oxidative damage, were measured in HAEC with or without vitamin C addition . The effect of vitamin C on eNOS activity was also determined . Results showed that HAEC without vitamin C treatment were essentially scorbutic . On addition of 100 mM vitamin C to the culture media, intracellular vitamin C levels increased and peaked at 6 h . A concomitant increase in the total GSH and the GSH/GSSG ratio was also observed; the NAD(P)H/NAD(P)+ ratio increased more slowly over the 24-h time course . Significantly lower (P <0.05) oxidant appearance and steady-state oxidative damage were also observed following vitamin C repletion . Vitamin C treatment increased eNOS activity by 600% . Thus, HAEC are scorbutic under normal culture conditions and exhibit higher oxidative stress than vitamin C repleted cells . Vitamin C supplementation should be considered when using cultured cells, especially when experimental endpoints are related to cellular redox status and eNOS activity.

Oncogene, 2002 Jun 13, 21(26), 4089 - 98
Maspin sensitizes breast carcinoma cells to induced apoptosis; Jiang N et al.; Maspin, a novel serine protease inhibitor (serpin), suppresses the growth and metastasis of breast tumor in vivo . However, the underlying molecular mechanism is unclear . In the current study, we report the first evidence that endogenous maspin expression in mammary carcinoma cells MDA-MB-435 enhanced staurosporine (STS)-induced apoptosis as judged by the increased fragmentation of DNA, increased proteolytic inactivation of poly-{ADP-ribose}-polymerase (PARP), as well as the increased activation of caspase-8 and caspase-3 . In parallel, recombinant maspin did not directly regulate the proteolytic activities of either caspase-3 or caspase-8 in vitro . Consistent with this result, maspin expressing normal mammary epithelial cells underwent more rapid STS-induced apoptosis as compared to breast carcinoma cells . Interestingly, maspin transfectant cells did not undergo spontaneous apoptosis in the absence of STS . Moreover, neither purified maspin protein added from outside nor endogenous maspin secreted to the cell culture media sensitized cells to STS-induced apoptosis . To investigate the structural determinants of maspin in its apoptosis-sensitizing effect, MDA-MB-435 cells were also transfected with maspin/PAI-1 and PAI-1/maspin chimeric constructs resulting from swapping the N-terminal and the C-terminal domains between maspin and PAI-1 (plasminogen activator inhibitor type 1) . The resulting stable transfectant clones expressing maspin/PAI-1 and PAI-1/maspin, respectively, did not undergo spontaneous apoptosis, and were similarly inhibited as maspin transfectant cells in motility assay . Interestingly, however, expression of both maspin/PAI-1 and PAI-1/maspin in MDA-MB-435 cells failed to sensitize these cells to STS-induced apoptosis . Taken together, our evidence provides new insights into the complex molecular mechanisms of maspin that may suppress breast tumor progression not only at the step of invasion and motility, but also by regulating tumor cell apoptosis . The sensitizing effect of maspin on apoptosis is to be contrasted by the pro-survival effect of several other serpins.

Blood, 2002 Jun 15, 99(12), 4443 - 8
Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth; Peyton KJ et al.; Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO) via the catabolism of heme by the enzyme heme oxygenase (HO) . In the present study, we found that serum stimulated a time- and concentration-dependent increase in the levels of HO-1 messenger RNA (mRNA) and protein in vascular SMCs . The induction of HO-1 expression by serum was inhibited by actinomycin D or cycloheximide . In addition, serum stimulated HO activity, as reflected by an increase in the concentration of bilirubin in the culture media . Treatment of vascular SMCs with serum stimulated DNA synthesis and this was potentiated by the HO inhibitors, zinc and tin protoporphyrin-IX as well as by the CO scavenger, hemoglobin . The iron chelator desferrioxamine had no effect on DNA synthesis . However, exposure of vascular SMCs to exogenous CO inhibited serum-stimulated SMC proliferation and the phosphorylation of retinoblastoma protein . In addition, CO arrested SMCs at the G(1)/S transition phase of the cell cycle and selectively blocked the serum-stimulated expression of cyclin A mRNA and protein without affecting the expression of cyclin D1 and E . CO also inhibited the serum-stimulated activation of cyclin A-associated kinase activity and cyclin-dependent kinase 2 activity . These results demonstrate that serum stimulates HO-1 gene expression and CO synthesis . Furthermore, they show that CO acts in a negative feedback fashion to inhibit vascular SMC growth by regulating specific components of the cell cycle machinery . The capacity of vascular mitogens to induce CO synthesis may provide a novel mechanism by which these agents modulate cell growth.

J Assist Reprod Genet, 2002 Apr, 19(4), 205 - 8
Successful elective single blastocyst transfer in a patient with prior repetitive high-order multiple gestations; Damario MA et al.; Multiple gestations remain one of the leading causes of morbidity related to infertility therapy . In the realm of assisted reproductive technologies, multiple gestations can be significantly limited by the reduction in the number of embryos transferred . Significant concern remains that a reduction in the number of embryos transferred may appreciably lower overall chances for pregnancy . Promising new developments are unfolding that may permit improved detection of a single human embryo with high implantation potential . One such development is the use of sequential culture media to allow prolonged culture of embryos to the blastocyst stage . We report a case in which sequential culture and elective transfer of one blastocyst was successfully used in a patient with a profoundly poor obstetrical history because of the complications of high-order multiple gestations.

Protein Eng, 2002 May, 15(5), 429 - 36
Interleukin-2-collagen chimeric protein which liberates interleukin-2 upon collagenolysis; Hayashi M et al.; Interleukin-2 (IL-2) is a potent activator of cellular immunity and has been utilized as an immunotherapeutic agent . We stably immobilized human IL-2 to collagen by covalently binding it to the N-terminus of human type III collagen (3A1) as IL2-3A1 chimeric protein using recombinant technology . The present study was aimed at liberating IL-2 from the immobilized chimeric protein by treating the chimera with bacterial collagenase . These IL2-3A1 chimeras were synthesized in insect cells which had been infected with baculovirus vectors carrying IL2-3A1 cDNA . The IL2-3A1 protein produced was shown to be in a pepsin-resistant triple helical structure and exhibited IL-2 activity to a similar extent as IL-2 itself . IL2-3A1 could be immobilized on the surface of plastic dishes by incubating it in the dishes . The IL-2 region of the immobilized IL2-3A1 was liberated to culture media by collagenase treatment and this freed IL-2 stimulated the growth of lined T cells . Thus, IL2-3A1 chimeric protein could be utilized as an IL-2 deliverer whose T cell mitogenic activity can be liberated by a collagenolytic environment.

Osteoarthritis Cartilage, 2002 May, 10(5), 344 - 52
Expression of TGF-betas and their receptors is differentially modulated by reactive oxygen species and nitric oxide in human articular chondrocytes; Ayache N et al.; OBJECTIVES: To study the effects exerted by two antioxidants, N-monomethyl-L-arginine (L-NMMA), as an inhibitor of nitric oxide (NO) synthesis, and N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, on the expression of the major growth factor involved in cartilage repair, TGF-beta, under the three isoforms beta1, beta2 and beta3, and the receptors I and II of this factor, using lipopolysaccharide (LPS)-treated human chondrocytes in culture . METHODS: Suspension cultures of human chondrocytes derived from the knee of osteoarthritic patients were treated for 48 h with lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5 mM) or NAC (1 mM) . Nitrite levels were assayed on the culture media using the Griess spectrophotometric method . After total RNA extraction, the expression of inducible NO synthase (iNOS), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta receptors I and II, was determined by semi-quantitative polymerase chain-reaction (RT-PCR) . RESULTS: LPS induced a dramatic increase of both NO production and iNOS mRNA level . The addition of L-NMMA (0.5 mM) abolished NO production without affecting iNOS mRNA levels . In contrast NAC (1 mM) strongly synergized with LPS to stimulate NO synthesis . LPS treatment did not significantly alter TGF-beta1 expression whereas L-NMMA inhibited its production . TGF-beta2 mRNA level was decreased by LPS and was not changed in the presence of L-NMMA . On the other hand, NAC was capable of counteracting the LPS-induced inhibition of TGF-beta2 expression . TGFbeta3 mRNA level was markedly reduced by LPS alone, or with both L-NMMA and NAC . Finally, the expression of TGF-betaRI was slightly increased in the presence of combined LPS and L-NMMA or NAC whereas that of TGFbeta-RII was reduced in the same conditions . CONCLUSIONS: The modulation of TGF-beta system was found to be differentially controlled by NO and ROS productions . Indeed, the control exerted on TGF-beta expression varied according to the isoform: TGF-beta1 mRNA level depends on NO whereas that of TGF-beta2 is regulated by ROS and TGF-beta3 seems to be unaffected by both of them . The expression of TGF-beta receptors appeared to be modulated by NO and ROS levels . The relevance of the present findings to osteoarthritis (OA) physiopathology and the potential use of antioxidant therapy to treat this disease are discussed .

Cytokine, 2002 Mar 7, 17(5), 254 - 61
Mechanism of inhibition of endothelin-1-stimulated proteoglycan and collagen synthesis in rat articular chondrocytes; Khatib AM et al.; The aim of this study was to determine the effects of endothelin-1 (ET-1) on proteoglycan (PG) and collagen synthesis by rat articular chondrocytes (RAC) . PG and collagen synthesis was measured by {(35)S}-sulphate and {(3)H}-glycine incorporation, respectively into monolayers of confluent RAC exposed to ET-1 (10(-11) M-10(-7) M) . ET-1 stimulated PG and collagen synthesis in these cells in a concentration-dependent manner during the first 24 h of incubation . Prolonged contact of the cells with ET-1 resulted in a gradual decrease, and finally, inhibition of ET-1 effects . This inhibition is mediated by nitric oxide (NO) released in response to ET-1 since: (1) nitric oxide synthase inhibitor, nitro-L-arginine-methyl ester (L-NAME), enhanced both basal and ET-1-induced {(35)S}-sulphate and {(3)H}-glycine incorporations; (2) sodium nitroprusside (SNP), which spontaneously releases NO, inhibited both basal and ET-1-induced incorporations, and was also able to suppress the effects of L-NAME; (3) NO levels in the culture media were also correlated with the inhibition of {(35)S}-sulphate and {(3)H}-glycine incorporation; and (4) SNP also inhibited aggrecan and collagen II transcriptions, probably via cGMP . This effect was mimicked with 8-bromo-cGMP . Interestingly, the LY83583, which blocks the NO-dependent production and release of cGMP, inhibited PG-collagen synthesis but had no effect on their mRNA expressions . Thus, normal levels of cGMP appeared to be necessary for PG-collagen synthesis, whereas decreased levels are detrimental . In conclusion, NO, produced by rat AC in response to ET-1, counteracts the stimulation and finally induces inhibition of PG-collagen synthesis by ET-1 in these cells but NO-induced cGMP is only partially responsible for this inhibition .

J Lab Clin Med, 2002 Apr, 139(4), 202 - 10
Dilinoleoylphosphatidylcholine prevents transforming growth factor-beta1-mediated collagen accumulation in cultured rat hepatic stellate cells; Cao Q et al.; Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines, protects against alcoholic and nonalcoholic liver fibrosis in baboons and rats, respectively . In this study, we assessed the antifibrogenic action of dilinoleoylphosphatidylcholine (DLPC), the main phosphatidylcholine species of PPC, against transforming growth factor-beta1-mediated expression of alpha1(I) procollagen, tissue inhibitor of metallopreoteinase-1 (TIMP-1) and matrix metalloproteinase-13 (MMP-13) in cultured rat hepatic stellate cells (HSCs) . In primary culture-activated HSCs, TGF-beta1 up-regulated the alpha1(I) procollagen mRNA level with a concomitant increase in type I collagen accumulation in culture media . Whereas TIMP-1 mRNA levels and TIMP-1 accumulation in media were also increased by TGF-beta1, MMP-13 mRNA expression and MMP-13 concentration in media were not altered . DLPC fully blocked TGF-beta1-induced increase in alpha1(I) procollagen mRNA expression and decreased collagen accumulation in media . Whereas TIMP-1 mRNA level and TIMP-1 accumulation in media were decreased by DLPC, MMP-13 mRNA expression and MMP-13 concentration in media were not changed by this treatment . Palmitoyl-linoleoylphosphatidylcholine (PLPC), the second most abundant component of PPC, had no effect on the concentrations of collagen, TIMP-1, and MMP-13 in HSC culture . We conclude that DLPC prevents TGF-beta1-mediated HSC fibrogenesis through down-regulation of alpha1(I) procollagen and TIMP-1 mRNA expression . The latter effect leads to a decreased accumulation of TIMP-1 that, in the presence of unchanged MMP-13 mRNA expression and MMP-13 concentration, results in a larger ratio of MMP-13/TIMP-1 concentrations in the culture media, favoring collagen degradation and lesser collagen accumulation . This effect of DLPC may explain, at least in part, the antifibrogenic action of PPC against alcoholic and other fibrotic disorders of the liver.

Ann Thorac Surg, 2002 May, 73(5), 1528 - 33
Use of autologous auricular chondrocytes for lining artificial surfaces: a feasibility study; Scott-Burden T et al.; BACKGROUND: Auricular elastic cartilage is a potential source of autologous cells for lining the luminal surfaces of cardiovascular prostheses . We tested this potential in vitro and in vivo using a left ventricular assist device (LVAD) and a calf model . METHODS: In vitro, auricular cartilage was harvested from the anesthetized ear of a calf, isolated, and cultured on tissue culture dishes . Primary chondrocytes were typed by immunocytochemistry, transferred into culture media, passaged twice, and seeded onto the blood-contacting luminal surfaces of four LVADs (HeartMate; Thoratec Corporation, Woburn, MA) . Seeded cell linings were preconditioned under simulated flow conditions to promote cell adhesion to luminal surfaces . Seeding efficiency and cumulative cell loss under flow conditions were quantitated . In vivo, one of the four autologous chondrocyte-lined and preconditioned LVADs was implanted into the tissue-donor calf; run for 7 days; explanted; and evaluated grossly, by scanning electron microscopy, and by transmission electron microscopy . RESULTS: The efficiency of seeding chondrocytes onto the luminal surfaces of the four LVADs was 95.11% +/- 4.23% (n = 4) . Cumulative cell loss during preconditioning under flow conditions in vitro did not exceed 12% (n = 4) . After 7 days of in vivo implantation, the luminal surfaces of the implanted LVAD demonstrated an intact, strongly adherent cellular lining . CONCLUSIONS: Auricular elastic cartilage is a ready and easily accessible source of chondrocytes whose ability to produce collagen II and other important extracellular matrix constituents allows them to adhere strongly to the luminal surfaces of LVADs . The simple method of isolating and expanding auricular chondrocytes presented here could be used to provide strongly adherent autologous cell linings for LVADs and other cardiovascular devices . If and when chondrocytes can be genetically engineered to produce antithrombogenic factors and then used to line the luminal surfaces of LVADs or other cardiovascular prostheses, they may be able to improve the hemocompatibility of the blood-biomaterial interface in such devices . Our successful feasibility study in a calf model warrants further studies of this concept in vivo.

Indian J Exp Biol, 2001 Dec, 39(12), 1243 - 8
Screening and in vitro production of diplodiatoxin from the isolates of Stenocarpella maydis and its toxigenic effect on bacterial strains; Rao SK et al.; Stenocarpella maydis from different maize growing regions in South Africa were collected and screened for the presence of diplodiatoxin . The presence of diplodiatoxin in these isolates was detected by thin layer chromatography and further confirmed by atomic pressure chemical ionization mass spectrometry . Samples containing diplodiatoxin showed a strong positive ion at m/z=307 . MC34, MC35, MC43 and MC50 isolates of Potchefstroom region produced high amount of diplodiatoxin, whereas some of the isolates from Potchefstroom (D72, D74, D78, D79 and D80) and Cedara (CH3 and U3H) regions did not contain diplodiatoxin . Experiments were conducted to optimize in vitro production of diplodiatoxin using the isolate MC 43 . A varied range of pH (3.0 to 5.0) and various culture media viz., PDB, CME, CLM and MSM were tested . Growth of mycelium and production of diplodiatoxin was maximum in PDB media at pH 4.5 and it was observed that diplodiatoxin was produced in detectable quantity in the cultures older than 6 weeks in this media . Further, diplodiatoxin was isolated and purified from 8-weeks-old cultures of MC43 isolate and confirmed by nuclear mass resolution . The standard and the compound purified showed similar NMR spectrum . Sixty-gram (fresh weight) mycelium yielded 19.52 mg of diplodiatoxin . Effect of diplodiatoxin on the growth of various bacterial strains in agar-gelled LB media was studied . They showed different range of tolerance to diplodiatoxin . The increasing order of tolerance to diplodiatoxin was Stenocarpella maydis < B . cereus < B . subtulus < P . fluorescense < E . coli . Further, the effect of different concentrations (4.88-49.70 microg/mL) of diplodiatoxin on the growth of S . aureus in LB liquid media was studied . Presence of diplodiatoxin in the media reduced cell growth as compared to the control thus, confirming anti-bacterial activity of diplodiatoxin.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 311 - 8
A technique for mycelial development of ectomycorrhizal fungi on agar media; De Araujo AA et al.; A technique was established to study ectomycorrhizal fungi on agar media . Petri dishes, 60 mm in diameter, containing 10 mL of culture medium covered with a cellophane disk were used for easy collection of the mycelium after growth . For analysis of fungal biomass production, a sterilized cellophane sheet was placed on the medium's surface . Inoculation was achieved by placing a mycelial block onto the center of the cellophane sheet and then incubating at 25 degrees C in the dark . Colony radial growth was measured and biomass dry wt was determined . Fresh mycelia were homogenized with 10 mL of acetate buffer (pH 5.5) for enzyme analysis . A crude extract was obtained by adding all culture medium to 90 mL of distilled water and homogenizing in a Potter . Reducing sugars, enzyme concentration, and pH were determined . Three fungal strains, Suillus collinitus, Pisosithus arrhizus, and Hebeloma cylindrosporum, were grown in different culture media (potato dextrose agar or Pintro's medium) . Parameters measured over time included glucose concentration, phosphatase activity, biomass, and pH.

J Helminthol, 2002 Mar, 76(1), 21 - 5
In vitro culture of tetrathyridia of Mesocestoides corti using a gel based diphasic medium; Chernin J et al.; Tetrathyridia of Mesocestoides corti were cultured in vitro in a diphasic medium consisting of a liquid medium (CMRL Sigma) and a thixotropic nutrient gel (Oxoid) . Tests demonstrated that a 50% medium/gel mixture produced optimum conditions for the survival and development of tetrathyridia . Established anthelminthic drugs were inoculated into the gel which demonstrated that this system can be used for preliminary anthelminthic drug screening . The development and survival of the tetrathyridia were influenced by the addition of pepsin, trypsin and liver peptone to the culture media . The development and maturation of proglottids were observed in addition to asexual reproduction by the process of budding . Tetrathyridia maintained in vitro and reinfected into both mouse and rat hosts retained their viability.

Biol Reprod, 2002 Jun, 66(6), 1672 - 80
Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone; Wang Y et al.; The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo . Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear . The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha) . In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth . The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization . Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth . The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM) . Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response . Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.

J Pathol, 2002 Jun, 197(2), 218 - 23
Expression of glycoprotein 90K in human malignant pleural mesothelioma: correlation with patient survival; Strizzi L et al.; The expression of the tumour-associated glycoprotein 90K in patients with malignant pleural mesothelioma (MM) has not been described . This study used enzyme-linked immunoassay (ELISA) to measure 90K in pleural effusions (PEs) and sera from patients with MM (n=28), lung cancer (LC) (n=14) and benign pleural disease (BPD) (n=15) . Immunohistochemistry was used to investigate 90K expression in MM and LC tissue sections . The expression of 90K was further evaluated in vitro by ELISA and western blot analysis of conditioned media and cellular extracts of MM, LC and normal human mesothelial (NHM) cell cultures . Finally, the relationships between 90K expression in MM and patient age and survival were studied . The mean 90K level was significantly higher (p<0.05) in PEs of MM patients (11.0+/-6.6 microg/ml) than in LC (6.1+/-3.2 microg/ml) or BPD (6.2+/-5.0 microg/ml) patients . Immunohistochemistry showed a positive reaction for 90K in MM biopsy sections and positive staining limited to inflammatory infiltrates in LC sections . The level of 90K was significantly higher in cell culture media of MM than of LC or NHM (p<0.001) . Bands representing proteins with molecular weight of approximately 90 kDa were detected by western blot in MM cellular extracts . An inverse correlation between PE 90K levels and MM patient age (r=-0.45; p=0.017) and a positive correlation between serum 90K levels and MM patient survival (r=0.62; p=0.006) were detected by linear regression analysis . Kaplan-Meier univariate analysis showed increased survival probability for MM patients with serum 90K level >7.3 microg/ml (log rank, p<0.05) . This is the first report in MM of the expression of 90K and of its potential diagnostic and prognostic application .

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Apr 25, 770(1-2), 111 - 9
Liquid chromatographic and electrophoretic characterisation of extracellular beta-glucosidase of Pleurotus ostreatus grown in organic waste; Morais H et al.; The production of beta-glucosidase by the ligninolytic fungus Pleurotus ostreatus has been studied in different culture media containing agro-industrial wastes . The enzyme is purified by anion-exchange chromatography, the molecular mass and isoelectric point of purified beta-glucosidase are measured by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing and the stability and kinetic parameters of the enzyme assessed by spectrophotometry . It has been established that the retention time, molecular mass and isoelectric point of the enzyme depend on the composition of the culture media while the activity and stability of beta-glucosidases of different origin were very similar . The combined chromatographic and electrophoretic methods have proved to be suitable techniques for the purification and characterisation of the beta-glucosidases produced by the ligninolytic fungus Pleurotus ostreatus in different culture media.

J Invertebr Pathol, 2001 Nov, 78(4), 201 - 9
Survival and differential development of Entomophaga maimaiga and Entomophaga aulicae (Zygomycetes: Entomophthorales) in Lymantria dispar hemolymph; Lopez Lastra CC et al.; The closely related entomophthoralean fungi Entomophaga aulicae and E . maimaiga are both host-specific pathogens of lepidopteran larvae . However, these fungi do not have the same host range . The first objective of this study was to compare the fate of E . aulicae in the nonpermissive host Lymantria dispar with the fate of the successful pathogen E . maimaiga over the same time period . In the hemolymph of L . dispar injected with E . maimaiga protoplasts, the number of hemocytes demonstrated a decreasing trend after the first day postinjection and hemocytes completely disappeared by day 5, with the majority of larvae dying in 5.6 +/- 0.1 days . In L . dispar larvae, E . maimaiga infections developed successfully, evidenced by increasing numbers of protoplasts and hyphal bodies prior to host mortality . In contrast, at day 5 hemocytes were readily visible in hemolymph of E . aulicae-injected larvae, but E . aulicae cells did not increase in numbers, although persisting in the hemolymph for at least 16 days postinjection . For both fungal species, when hemolymph samples from injected insects were introduced to culture media viable fungal cultures were always produced . Both E . aulicae and E . maimaiga occurred in hemolymph initially after injection as protoplasts . For E . maimaiga, after day 3, <50% of fungal cells were hyphal bodies until insect death when most cells regenerated cell walls . For E . aulicae, from day 2 equal numbers of fungal cells in the hemolymph occurred as protoplasts and hyphal bodies . To investigate the cause of fungistasis in E . aulicae-injected larvae, E . aulicae cell cultures exposed to partially purified protein fractions from hemolymph of larvae infected with either fungus displayed increased lysis and decreased viability at lower concentrations of protein fractions compared with E . maimaiga cell cultures . These studies demonstrate that E . aulicae does not increase in L . dispar hemolymph, although it persists and results suggest that proteinaceous factors induced within the hemolymph may limit the capacity of E . aulicae to develop successful infections.

J Bone Miner Res, 2002 May, 17(5), 774 - 81
Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines; Ono K et al.; We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear . In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines . Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner . Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562 . The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured . The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA . In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs . Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae . Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein . These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.

Glia, 2002 Jun, 38(4), 292 - 302
Astrocyte-derived factors modulate the inhibitory effect of ethanol on dendritic development; Yanni PA et al.; Numerous studies in vivo and in vitro have demonstrated that ethanol disrupts neuromorphogenesis . However, it has not been determined what role, if any, is played by non-neuronal cells in mediating this effect . We recently reported that ethanol inhibits dendritic development in low-density cultures of fetal rat hippocampal pyramidal neurons (Yanni and Lindsley, 2000: Dev Brain Res 120:233-243) . In this culture system, cortical astrocytes precondition neuronal culture media for 2 days before the addition of neurons, which then develop on a separate substrate in coculture with the astrocytes . To determine whether astrocyte response to ethanol mediates the effects of ethanol on neurons, the present study compared dendritic development of neurons after 6 days in medium containing 400 mg/dl ethanol in coculture with live astrocytes and in conditioned medium from astrocytes that were never exposed to ethanol . The same experiment was also performed with and without ethanol present during astrocyte preconditioning of the medium . The effects of ethanol differed depending on when it was added to the cultures relative to addition of newly dissociated neurons . However, the effects of ethanol were not related to whether neurons were cocultured with live astrocytes . When astrocytes preconditioned the medium normally, ethanol added at plating inhibited dendritic development of neurons regardless of whether they were maintained in coculture with live astrocytes or in conditioned medium . In surprising contrast, the presence of ethanol during astrocyte preconditioning of the media had a growth promoting effect on subsequent dendrite development despite the continued presence of ethanol in the medium . Thus, astrocytes release soluble factors in response to ethanol that can protect neurons from the inhibitory effects of ethanol on dendritic growth, but the timing of neuronal exposure to these factors, or their concentration, may influence their activity .

J Assist Reprod Genet, 2002 Mar, 19(3), 137 - 43
Protein supplementation of human IVF culture media; Blake D et al.; This review travels the road of protein supplementation in embryo culture development-from whole crude plasma in the mid Twentieth century moving through to the completely genetically engineered human albumin with successful births at the beginning of the Twenty-first.

Histochem J, 2001 Sep-Oct, 33(9-10), 553 - 8
Hyaluronan digestion and synthesis in an experimental model of metastatic tumour; Delpech B et al.; To approach the question of hyaluronan catabolism in tumours, we have selected the cancer cell line H460M, a highly metastatic cell line in the nude mouse . H460M cells release hyaluronidase in culture media at a high rate of 57 pU/cell/h, without producing hyaluronan . Hyaluronidase was measured in the H460M cell culture medium at the optimum pH 3.8, and was not found above pH 4.5, with the enzyme-linked sorbent assay technique and zymography . Tritiated hyaluronan was digested at pH 3.8 by cells or cell membranes as shown by gel permeation chromatography, but no activity was recorded at pH 7 with this technique . Hyaluronan was digested in culture medium by tumour slices, prepared from tumours developed in nude mice grafted with H460M cells, showing that hyaluronan could be digested in complex tissue at physiological pH . Culture of tumour slices with tritiated acetate resulted in the accumulation within 2 days of radioactive macromolecules in the culture medium . The radioactive macromolecular material was mostly digested by Streptomyces hyaluronidase, showing that hyaluronan was its main component and that hyaluronan synthesis occurred together with its digestion . These results demonstrate that the membrane-associated hyaluronidase of H460M cells can act in vivo, and that hyaluronan, which is synthesised by the tumour stroma, can be made soluble and reduced to a smaller size by tumour cells before being internalised and further digested.

Microbiol Res, 2002, 157(2), 115 - 25
Diversity in soil fungi from undisturbed and disturbed Celtis tala and Scutia buxifolia forests in the eastern Buenos Aires province (Argentina); Cabello M et al.; The rhizospheric soil microfungi from a native forest (undisturbed and disturbed) were studied using soil dilution plate and soil washing methods . Fungi were isolated using slightly acid and alkaline culture media . 54 taxa were isolated: 49 from undisturbed forest soil and 37 from disturbed forest soil . Acremonium sp., Aspergillus ustus, Coemansia pectinata, Doratomyces stemonitis, Fusarium solani, F . oxysporum, Gliocladium roseum, Humicola fusco-atra, Mortierella sp., Penicillium lilacinum, Trichoderma harzianum, and T koningii, showed the highest frequency, in both, undisturbed and disturbed forests . In undisturbed soil forest the biodiversity index was 3.97 whereas in disturbed ones was 3.89.

Clin Oral Investig, 2002 Mar, 6(1), 39 - 50
Human dermal and gingival fibroblasts in a three-dimensional culture: a comparative study on matrix remodeling; Chaussain Miller C et al.; Free-floating collagen lattice is considered a useful tool for assessing wound healing in vitro . This work compared extracellular matrix remodeling in collagen lattices populated by gingival or dermal fibroblasts . For 21 days we followed gel contraction and changes in cell number of collagen lattices seeded with l.5 x 10(5) fibroblasts of each tissue . We also used indirect immunodetection to study extracellular matrix components, metalloproteinases (MMPs), and their tissues inhibitors (TIMPs) . In addition, the presence of MMPs and TIMPs in the culture media was analyzed by zymography and western blotting . No significant difference was found concerning gel contraction and changes in cell number . We observed the early expression of fibrillin I and collagen type III, apparently codistributed and at the end of the gel contraction their disappearance . Concomitantly we demonstrated the expression of MMPs and TIMPs, initially localized in cellular cytoplasm, then spreading in the extracellular compartment, and even found in the culture medium . This remodeling was more rapid and intense with gingival fibroblasts than dermal fibroblasts . In conclusion, gingival fibroblasts seem more efficient at remodeling the connective tissue than dermal fibroblasts and could lead to the better wound healing observed in vivo.

Clin Appl Thromb Hemost, 2002 Jan, 8(1), 65 - 71
Effects of a low molecular weight heparin, bemiparin, and unfractionated heparin on hemostatic properties of endothelium; Perez-Ruiz A et al.; Human endothelial cells synthesize and secrete a variety of molecules involved in fibrinolysis and coagulation . The effects of a low molecular weight heparin, Bemiparin, and unfractionated heparin (UFH) were compared on plasminogen activator inhibitor-1 (PAI-1), tissue-plasminogen activator (t-PA), tissue factor (TF), tissue factor pathway inhibitor (TFPI) release, and PAI-1 gene expression by human umbilical vein endothelial cells (HUVEC) . Cell cultures were supplemented with Bemiparin or UFH at 1 or 10 U/mL . Culture media samples were obtained before the addition of the drugs and 2, 6, and 24 hours afterward to measure the antigen levels of TF, TFPI, t-PA, and PAI-1 . RNA was obtained to study the endothelial expression of PAI-1 by reverse transcriptase-polymerase chain reaction (RT-PCR) . Bemiparin at 1 U/mL resulted in a decreased messenger RNA (mRNA) PAI-1 expression, which remained unaltered when UFH had been added . PAI-1 levels increased after the cultures had been supplemented with either Bemiparin or UFH at both doses . UFH induced an increase in t-PA either at 1 or 10 U/mL . Both doses of UFH, but not Bemiparin, induced an important increase in TF secretion . An increase in the TFPI levels was seen with UFH at 1 U/mL . The decrease in PAI-1 gene expression observed with a therapeutic dose of Bemiparin might confer this drug interesting profibrinolytic properties . The fact that Bemiparin, in contrast with UFH, does not induce an increase in TF could give this drug another positive feature.

Scand J Gastroenterol, 2002 Apr, 37(4), 467 - 75
Effects of platelet activating factor, butyrate and interleukin-6 on cyclooxygenase-2 expression in human esophageal cancer cells; Wang LS et al.; BACKGROUND: Epidemiological studies have indicated that non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of esophageal squamous cell carcinoma (ESCC) by taking cyclooxygenase (COX) as the target enzyme . The pathophysiological regulation of COX-2 may play a role in carcinogenesis and in disease progression of esophageal carcinoma . METHODS: 59 ESCC samples were used to assess COX-2 expression in the tumor cells and four ESCC cell lines to investigate the effects of phorbol myristate acetate (PMA), platelet activating factor (PAF), n-sodium butyrate (n-BT) and interleukin-6 (IL-6) on the expression of COX-2 . Expression of COX-2 was determined by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) . Production of PGE2 was measured by a competitive enzyme immunoassay (CEIA) . RESULTS: COX-2 expression was detected in 54.2% (32/59) of the pathological sections by IHC . COX-2 expression in ESCC cells was significantly increased following treatment with PAF and n-BT . Increased production of PGE2 was detected in the culture media, and the secreted PGE2 in the culture media was proportional to the increased COX-2 expression . The addition of IL-6 could also enhance COX-2 expression in ESCC cells . While NSAIDs could inhibit enzymatic activity of COX-2, they did not inhibit COX-2 gene expression in ESCC cells . PKC inhibitor, however, could abrogate PMA-induced COX-2 gene expression, but it did not block IL-6-induced COX-2 expression . CONCLUSIONS: Our data suggest that COX-2 expression in ESCC cells could be upregulated by PMA, PAF, n-BT and IL-6 . Nonetheless, IL-6-induced COX-2 expression could be independent of PKC activation.

Blood, 2002 May 15, 99(10), 3654 - 60
Analysis of fibrinogen gamma-chain truncations shows the C-terminus, particularly gammaIle387, is essential for assembly and secretion of this multichain protein; Okumura N et al.; To examine the role of the fibrinogen gamma chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated gamma chains, terminating between residues gamma379 and the C-terminus, gamma411 . The variant fibrinogens were synthesized from altered gamma-chain complementary DNAs in cultured Chinese hamster ovary cells . Immunoassays of the culture media demonstrated that only those variants with gamma chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the gamma chain, from 1.4 microg/mL for normal fibrinogen to 0.39 microg/mL for gamma 387 fibrinogen . Immunoassays of cell lysates showed that all variant gamma chains were synthesized, although the levels varied significantly . For variants longer than 386 residues, levels decreased with length but remained near normal . In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced . Quantitative reverse transcription-polymerase chain reaction demonstrated that the gamma-chain messenger RNA level was independent from chain length . Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules . For shorter variants, these intermediates were not evident . We conclude that residues near the C-terminus of the gamma chain are essential for fibrinogen assembly, and more specifically, that gamma387 is critical . We propose that the loss of residue gamma387 destabilized the structure of gamma chain, preventing assembly of alphagamma and betagamma dimers, essential intermediates in the assembly of normal fibrinogen.

Hum Reprod, 2002 May, 17(5), 1306 - 10
Embryonic platelet-activating factor: an indicator of embryo viability; Roudebush WE et al.; BACKGROUND: A definitive need exists to identify a biomarker of embryonic viability . Platelet-activating factor (PAF) production by human embryos is related to pregnancy potential . METHODS: Conditioned embryo culture media were obtained following conventional IVF on day 3, with PAF levels and pregnancy outcomes correlated . RESULTS: Overall pregnancy rate was 68% (17/25) with a mean of 84.1 (+/- 8.5) pmol/l/embryo PAF level . PAF levels ranged from a 216.4 pmol/l/embryo (pregnant) to a 3.7 pmol/l/embryo (not pregnant) . There was a significant difference (P < 0.05) in PAF content between pregnant (92.1 +/- 9.5 pmol/l/embryo) and non-pregnant groups (52.5 +/- 16.6 pmol/l/embryo) . Patients were categorized into three groups based upon PAF levels: low (< or= 5 pmol/l/embryo); medium (51-100 pmol/l/embryo) and high (>100 pmol/l/embryo) . The low (60%) group had a significantly (P < 0.05) lower pregnancy rate than either the medium (85%) or high (89%) groups . A receiver-operator characteristic curve predicted a cut-off limit of 45 pmol/l/embryo for PAF content in human embryo conditioned culture media . CONCLUSIONS: The data demonstrate a correlation between PAF levels in human embryo conditioned culture media and pregnancy outcome . Additionally, as embryonic PAF levels increase so does the corresponding pregnancy rate . Therefore, PAF may be used as an indicator of embryo viability and for predicting pregnancy outcome.

Reprod Suppl, 2001, 58, 159 - 73
In vitro fertilization and embryo development in pigs; Abeydeera LR; Considerable progress has been made in the in vitro production of pig embryos using improved methods for in vitro maturation (IVM) and fertilization (IVF) . Despite the progress, polyspermic penetration remains a problem for in vitro-matured oocytes . Variation among boars, ejaculates and IVF protocols used in different laboratories appears to influence the incidence of polyspermy . Recent studies indicate that oviduct cells and their secretions play a role in reducing polyspermy . Very early attempts to culture in vivo-derived pig embryos met with little success and most were arrested at the four-cell stage . At present, many culture media are available that can overcome the four-cell block and support development to the blastocyst stage . In contrast, blastocyst development of in vitro-produced (IVP) embryos in these culture media varies significantly . Significant differences in morphology and numbers of cells have been observed in in vitro-produced blastocysts compared with in vivo-derived blastocysts . Surgical transfer of in vitro-produced embryos to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes . Although several systems are available for the generation of in vitro-produced embryos, the problems of polyspermy and poor embryo survival prevent large-scale production of embryos . Further research should be directed to improve oocyte and embryo quality, and to develop methods to minimize polyspermy through development of better IVM, IVF and embryo culture techniques.

Am J Dermatopathol, 2002 Apr, 24(2), 118 - 29
Anetoderma: an altered balance between metalloproteinases and tissue inhibitors of metalloproteinases; Ghomrasseni S et al.; The amount of elastic fibers from lesional and healthy skin areas of five patients with anetoderma was determined by automated image analysis . Dermal elastic fibers were almost completely absent in anetodermic skin and preelastic fibers were undetectable or extremely rare . Organ cultures were performed using explants from affected and unaffected skin areas of the same patient . We identified and quantified proteases in the culture media of explants: MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MMP-3 (stromelysin 1), MMP-7 (matrilysin 1), and tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2 . The data were compared with those of two healthy donors . For the five samples of anetodermic skin, MMP-1 levels were significantly higher compared with the uninvolved cultures and the two healthy samples . A significant increase of TIMP-1 expression was also observed in the affected cultures . We demonstrated a significant increase in the production of gelatinase A in lesional skin when compared with nonlesional skin and healthy donor samples . We found no significant production of TIMP-2 in the five samples of anetodermic skin compared with the samples from the two healthy donors . There was a significant decrease in TIMP-2 expression in the five nonlesional samples compared with the control samples . These data are in favor of an altered balance in anetodermic patients between MMP-2 and TIMP-2 . Levels of MMP-9, MMP-3, and MMP-7 were significantly higher in the culture-conditioned media of the anetodermic skin samples than the nonlesional skin cultures . Because MMP-3, MMP-7, MMP-9 are known to degrade elastin, and MMP-3 can activate the latent forms of MMP-7 and MMP-9, we propose that these metalloproteinases also participate in the degradation of elastic fibers in anetodermic skin.

Diabetes, 2002 May, 51(5), 1398 - 408
Combined expression of pancreatic duodenal homeobox 1 and islet factor 1 induces immature enterocytes to produce insulin; Kojima H et al.; Immature rat intestinal stem cells (IEC-6) given the ability to express the transcription factor, pancreatic duodenal homeobox 1 (Pdx-1), yielded YK cells . Although these cells produced multiple enteroendocrine hormones, they did not produce insulin . Exposure of YK cells to 2 nmol/l betacellulin yielded BYK cells that showed the presence of insulin expression in cytoplasm and that secreted insulin into culture media . By examining the mechanism of differentiation in BYK cells, we found that another transcription factor, islet factor 1 (Isl-1) was newly expressed with the disappearance of Pax-6 expression in those cells after exposure to betacellulin . These results indicated that combined expression of Pdx-1 and Isl-1 in IEC-6 cells was required for the production of insulin . In fact, overexpression of both Pdx-1 and Isl-1 in IEC-6 cells (Isl-YK-12, -14, and -15 cells) gave them the ability to express insulin without exposure to betacellulin . Furthermore, implantation of the Isl-YK-14 cells into diabetic rats reduced the animals' plasma glucose levels; glucose levels dropped from 19.4 to 16.9 mmol/l 1 day after the injection of cells . As expected, the plasma insulin concentrations were 2.7 times higher in the diabetic rats injected with Isl-YK-14 cells compared to in controls . In summary, our results indicated that immature intestinal stem cells can differentiate into insulin-producing cells given the ability to express the transcription factors Pdx-1 and Isl-1.

Altern Lab Anim, 2002 Mar-Apr, 30(2), 219 - 27
The use of fetal bovine serum: ethical or scientific problem?
Jochems CE, van der Valk JB, Stafleu FR, Baumans V.
Fetal bovine serum (FBS) is a common component of animal cell culture media . It is harvested from bovine fetuses taken from pregnant cows during slaughter . FBS is commonly harvested by means of a cardiac puncture without any form of anaesthesia . Fetuses are probably exposed to pain and/or discomfort, so the current practice of fetal blood harvesting is inhumane . Apart from moral concerns, several scientific and technical problems exist with regard to the use of FBS in cell culture . Efforts should be made to reduce the use of FBS or, preferably, to replace it with synthetic alternatives.

Biol Reprod, 2002 May, 66(5), 1380 - 6
Conditions for in vitro maturation and artificial activation of ferret oocytes; Li Z et al.; The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time . In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined . Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG {10 IU/ml} and hCG {5 IU/ml}), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol {2 microg/ml}) . After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73) . At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media . For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination . Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage . Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59) . Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells . These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.

Surg Endosc, 2002 Jan, 16(1), 36 - 9 Epub 2001 Oct 19.
Higher colon cancer tumor proliferative index and lower tumor cell death rate in mice undergoing laparotomy versus insufflation; Lee SW et al.; BACKGROUND: Our laboratory has previously shown that tumors are established more easily and grow larger after laparotomy than after laparoscopy . To characterize these differences in tumor growth further, the tumor cell death rates and tumor proliferation rates were compared in vivo after full sham laparotomy versus carbon dioxide (CO2) insufflation . METHODS: Female Balb/C mice (n = 36) were inoculated intradermally in the dorsal skin with 106 C-26 colon adenocarcinoma cells in 0.1 ml of culture media no more than 1 h before interventions . The mice then were randomized to one of three groups: anesthesia control, CO2 insufflation, or sham laparotomy . The anesthesia control group underwent no procedure . The insufflation group underwent CO2 pneumoperitoneum (4-6 mmHg) for 20 min via a 20-gauge angiocatheter . The laparotomy group underwent a midline incision from xiphoid to pubis, which was closed after 20 min . Tumors were excised from half the mice in each group on postoperative day 7, and from the remaining mice on postoperative day 14 . Sections of tumors were made then stained separately for free 3? hydroxyl ends of genomic deoxyribonucleic acid (DNA) using fluorescein-deoxyunidine triphosphate (dUTP), and immunohistochemically for proliferating cell nuclear antigen (PCNA) . Apoptosis was measured by quantitating DNA strand breaks in individual cells using fluorescence microscopy . Fluorescein-positive cells in five random high-power fields (x200) were counted in a blinded fashion . The proliferative index of each tumor was determined by averaging PCNA positive cells in five high-power fields (x450) counted in a blinded fashion with the aid of an optical grid . RESULTS: On postoperative day 7, there was no significant difference in the proliferative index or apoptotic rates among the three groups . On postoperative day 14, the proliferative index in the laparotomy group was significantly higher than in either the insufflation or control group (p < 0.001) . The proliferative index in the insufflation group also was significantly higher than in the control group (p < 0.05) . Inverse differences in apoptotic rates were found . The apoptotic rate in the laparotomy group was significantly lower than in either the insufflation (p < 0.05) or control group (p < 0.001) . The apoptotic rate in the insufflation group was significantly lower than in the control group (p < 0.001) . CONCLUSIONS: We have demonstrated that there is a significantly higher rate of tumor cell proliferation and a significantly lower rate of tumor cell death with the C-26 colon adenocarcinoma tumor line after laparotomy than after insufflation or anesthesia alone on post-operative day 14 . The mechanisms of these phenomena are unclear . It appears that certain factors postoperatively stimulate tumors to proliferate at a higher rate, causing tumor cells to die at a lower rate in the laparotomy group than in the insufflation group.

Gynecol Obstet Invest, 2002, 53(2), 105 - 11
Human endometrial epithelial cells modulate the activation of gelatinase a by stromal cells; Goffin F et al.; Metalloproteinases (MMPs) are central effectors in endometrial physiology . Their production is tightly regulated by ovarian steroids and cytokines . Using zymography, we investigated MMP-2 production by human endometrial cells treated with estradiol-17beta + progesterone (E(2)+P) and by various key cytokines in endometrial physiology (IL-1beta, LIF, TGF-beta, and TNF-alpha) . No gelatinase activity was detected in the culture media of epithelial cells . In basal conditions, stromal cells produced the pro form of MMP-2 . MMP-2 production/activation was not directly affected by cytokine treatment . Interestingly, activated MMP-2 was only detected after treatment of stromal cells with culture medium from epithelial cells . Cytokine treatment of epithelial cells increased the capacity of conditioned medium to stimulate stromal cells to activate MMP-2 . As the tissue inhibitor of MMP-2 (TIMP-2) is a regulator of gelatinase A activity, its concentration was measured by ELISA . TIMP-2 production by stromal cells was not affected by cytokines or by epithelial cell-conditioned medium . These results strongly suggest that regulation of stromal MMP-2 activation involves soluble factor(s) derived from the epithelial compartment .

Toxicol Sci, 2002 May, 67(1), 32 - 7
In vitro bioavailability of heavy metals in pressure-treated wood dust; Gordon T et al.; Pressure treatment with chromium, copper, and arsenic (CCA) is the most prevalent method for protecting wood used in outdoor construction projects . Although these metals are tightly bound to the wood fibers and are not released under most conditions of use, we examined the bioavailability of metals in CCA pressure-treated wood dust in vitro . Cytotoxicity and metallothionein (MT) mRNA expression were examined in V79 Chinese hamster lung fibroblast cells incubated with respirable-size wood dust generated by sanding CCA-treated and untreated (control) Southern yellow pine . In colony survival studies, increased cytotoxicity (p < 0.05) occurred in V79 cells treated with CCA wood dust (351 +/- 77 microg/ml, mean +/- SE) compared with control wood dust (883 +/- 91 microg/ml) . Increased cytotoxicity with CCA wood dust also occurred in an arsenic resistant subline of V79 cells, thus suggesting that arsenic was not responsible for the increased cytotoxicity . Metallothionein mRNA was significantly increased after 48 h of treatment with CCA wood dust compared with control wood dust . Incubation of CCA wood dust in cell culture media resulted in the transfer of copper, but not chromium or arsenic, into the media . Moreover, the treatment of cells with this filtered extract resulted in significantly increased metallothionein mRNA, suggesting that bioavailable copper is responsible for inducing metallothionein mRNA in V79 cells . Thus, these bioassays suggest that metals become bioavailable during in vitro culture of phagocytic V79 cells with CCA wood dust.

Endocrinology, 2002 May, 143(5), 1922 - 31
Leptin promotes the development of mouse preimplantation embryos in vitro; Kawamura K et al.; Leptin acts as a modulator of diverse reproductive functions, and recent studies have implicated involvement of leptin in the early embryo development in mammal . The aim of this study was to investigate the expression of leptin and its receptor (OB-R) in mouse oocyte and preimplantation embryo, and to examine whether leptin influenced the early embryo development . Leptin mRNA was detected in blastocyst and hatched blastocyst, and two splice variants of OB-R (OB-Ra and OB-Rb) mRNAs were detected in oocytes, 1-cell, 2-cell, morula, blastocyst, and hatched blastocyst . As for the origin of leptin, both leptin mRNA and protein were identified in the oviduct epithelium and endometrium of pregnant mouse . In the pregnant mouse, the levels of leptin in uterine fluid were higher than those in nonpregnant mouse . Addition of leptin to embryo culture media promotes the development from 2-cell stage embryos to the blastocysts, fully expanded blastocysts and hatched blastocysts . This effect was neutralized by an antibody against the extracellular domain of OB-R . Leptin significantly increased the total cell number of blastocysts, and the effect was preferentially observed in the trophectoderm . These findings raise the possibility of a paracrine/autocrine leptin signaling system regulating the development of mouse preimplantation embryo.

Graefes Arch Clin Exp Ophthalmol, 2002 Jan, 240(1), 42 - 8
Inhibitory effects of triamcinolone acetonide on bFGF-induced migration and tube formation in choroidal microvascular endothelial cells; Wang YS et al.; BACKGROUND: Angiostatic drugs might provide desirable modulation of choroidal angiogenesis-related diseases, including histoplasmosis and the exudative form of age-related macular degeneration . However, the precise effects of this class of compounds in the choroidal neovascularization are still unclear . In the present study, we investigated the effects of triamcinolone acetonide (TA), an angiostatic steroid, on choroidal angiogenesis in vitro . METHODS: Bovine choroidal endothelial cells (CEC), which are the critical cellular component of choroidal angiogenesis in vivo, were isolated with Lycopersicon esculentum agglutinin-coated Dynabeads and cultured in EGM medium . CEC were treated with basic fibroblast growth factor (bFGF) and TA at various concentrations ranging from 50 to 300 mg/l . The capacities for CEC migration and tube formation were evaluated with the modified Boyden chamber and the Vitrogen collagen assay, respectively . The activities of matrix metalloproteinases (MMP)-2 and -9 were examined using gelatin zymography . RESULTS: The stimulation of CEC with 50 ng/ml bFGF resulted in an increase of about 100% in migration activity (P<0.01) . Preincubation of CEC with TA at the indicated concentrations for 20 min inhibited the bFGF-stimulated migration in a dose-dependent manner (P<0.01) . After 5 days, the bFGF-stimulated tube formation in CEC was inhibited by TA at the concentrations 100, 150 and 300 mg/l (P<0.01) . Gelatin zymography of the culture media of CEC showed that the bFGF-induced activation of MMP-2 was attenuated by 300 mg/l TA (P<0.05) . CONCLUSION: Downregulation of the activation of MMPs in CEC could be one of the mechanisms by which angiostatic steroids inhibit choroidal angiogenesis.

Arthritis Rheum, 2002 Apr, 46(4), 968 - 75
Influence of hypoxia and reoxygenation on cytokine-induced production of proinflammatory mediators in articular cartilage; Cernanec J et al.; OBJECTIVE: Articular cartilage is an avascular tissue that functions at a lower oxygen tension than do most tissues . With mobilization, arthritic joints may undergo cycles of hypoxia and reoxygenation . The goal of this study was to determine the effects of hypoxia and reoxygenation on cytokine-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in articular cartilage . METHODS: Porcine cartilage explants were incubated at 37 degrees C for 72 hours in either 1% O(2) (hypoxia) or 20% O(2) (normoxia) in media supplemented with interleukin-1alpha (IL-1alpha) or tumor necrosis factor alpha (TNFalpha), with or without the NO synthase 2 (NOS2) selective inhibitor 1400W . Culture media were then removed and replaced with freshly prepared media and incubated for a further 24 hours in normoxia . RESULTS: NO levels were significantly higher in explants supplemented with IL-1alpha and TNFalpha compared with controls, in both hypoxia and normoxia . Compared with normoxia, hypoxia decreased IL-1alpha- and TNFalpha-induced NO production significantly . Reoxygenation of hypoxic explants resulted in sustained significant NO production in response to either cytokine . However, comparably high levels of NO production were not sustained in explants cultured continuously in normoxia . Although IL-1alpha alone did not significantly increase PGE(2) production, significant PGE(2) superinduction occurred in cartilage stimulated with IL-1alpha and the NOS2 inhibitor 1400W compared with stimulation with IL-1alpha alone in hypoxia, but not in normoxia . CONCLUSION: Oxygen tension significantly affects cytokine-induced proinflammatory mediator production in articular cartilage . Furthermore, hypoxia alters NO mediation of PGE(2) production . Hypoxia and reoxygenation can affect cytokine-induced proinflammatory mediator production, suggesting that oxygen tension may influence inflammation associated with cartilage injury and disease.

Zhonghua Fu Chan Ke Za Zhi, 2002 Mar, 37(3), 152 - 4
{Study of effects of leptin on cultured human luteinized granulosa cell function}; Huang H et al.; OBJECTIVE: To assess the effects of leptin on follicle-stimulating hormone (FSH) and insulin-like growth factor-I (IGF-I) induced production of estradiol (E(2))-17beta and progesterone (P) in cultured human luteinized granulosa cell (GC) . METHODS: Human luteinized GCs were obtained from pre-ovulatory follicles in an in-vitro fertilization program, and were cultured for 24 hours in the presence of leptin (3.0 ng/ml) or FSH (1.0 ng/ml) or IGF-I (30.0 ng/ml) alone, leptin with FSH or IGF-I or both, and FSH with IGF-I . The conditioned media were aspirated for measuring E(2) and P content . Luteinized GC were also counted for cell number and detected the expression of the leptin receptor by reverse transcription polymerase chain reaction analysis . RESULTS: In cultured luteinized GC system, leptin or FSH or IGF-I alone did not affect the growth of cultured human luteinized GC . Leptin alone or in the presence of FSH had no effect on E(2) production . E(2) levels in culture media without leptin or FSH were (0.103 +/- 0.036) pmol/1 000 cells, (0.323 +/- 0.042) pmol/1 000 cells respectively . When cultured with leptin alone or with leptin and FSH, E(2) levels were (0.120 +/- 0.008) pmol/1 000 cells, (0.343 +/- 0.034) pmol/1 000 cells . Leptin significantly inhibited FSH + IGF-I or IGF-I induced E(2) production . E(2) levels decreased from (0.318 +/- 0.037) pmol/1 000 cells to (0.193 +/- 0.025) pmol/1 000 cells (IGF-I) (P < 0.05) and from (0.493 +/- 0.036) pmol/1 000 cells to (0.251 +/- 0.033) pmol/1 000 cells (FSH + IGF-I) (P < 0.01) . However, the production of P did not change . The leptin receptor expression was demonstrated in luteinized GC . CONCLUSIONS: Leptin may directly attenuate the IGF-I or IGF-I and FSH-stimulated E(2) synthesis in cultured human luteinized GC by the leptin receptor mechanism.

Arterioscler Thromb Vasc Biol, 2002 Apr 1, 22(4), 566 - 73
Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia; Pearlstein DP et al.; Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers . These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium . We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia . Oxidation of the probe 2', 7'-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance . Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 micromol/L) and diphenyleneiodonium (5 micromol/L) . The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-kappaB (NF-kappaB) activation, although they did not abrogate NF-kappaB activation in response to endotoxin (lipopolysaccharide) . ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers . By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 micromol/L), the xanthine oxidase inhibitor allopurinol (100 micromol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 micromol/L) . We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-kappaB activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia.

Osteoarthritis Cartilage, 2002 Apr, 10(4), 308 - 20
Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors; Ellsworth JL et al.; OBJECTIVE: The aim of this study was to examine the effects of recombinant human Fgf18 on chondrocyte proliferation and matrix production in vivo and in vitro . In addition, the expressions of Fgf18 and Fgf receptors (Fgfr) in adult human articular cartilage were examined . METHODS: Adenovirus-mediated transfer of Fgf18 into murine pinnae and addition of FGF18 to primary cultures of adult articular chondrocytes were used to assess the effects of FGF18 on chondrocytes . In situ hybridization was used to examine the expression of Fgf18 and Fgfr s in adult human articular cartilage . RESULTS: Expression of Fgf18 by adenovirus-mediated gene transfer in murine pinnae resulted in a significant increase in chondrocyte number . Chondrocytes were identified by staining with toluidine blue and a monoclonal antibody directed against type II collagen . Fgf18, Fgfr 2-(IIIc), Fgfr 3-(IIIc), and Fgfr 4 mRNAs were detected within these cells by in situ hybridization . The nuclei of the chondrocytes stained with antibodies to PCNA and FGF receptor (FGFR) 2 . Addition of FGF18 to the culture media of primary articular chondrocytes increased the proliferation of these cells and increased their production of extracellular matrix . To assess the receptor selectivity of FGF18, BaF3 cells stably expressing the genes for the major splice variants of Fgfr1-3 were used . Proliferation of cells expressing Fgfr 3-(IIIc) or Fgfr 2-(IIIc) was increased by incubation with FGF18 . Using FGFR-Fc fusion proteins and BaF3 cells expressing Fgfr 3-(IIIc), only FGFR 3-(IIIc)-Fc, FGFR 2-(IIIc)-Fc or FGFR 4-Fc reduced FGF18-mediated cell proliferation . Expression of Fgf18, Fgfr 3-(IIIc) and Fgfr 2-(IIIc) mRNAs was localized to chondrocytes of human articular cartilage by in situ hybridization . CONCLUSION: These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro . Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage .

J Dairy Sci, 2002 Mar, 85(3), 562 - 9
Protective effect of melatonin and catalase in bovine neutrophil-induced model of mammary cell damage; Boulanger V et al.; The effect of several antioxidants and a proteinase inhibitor on bovine neutrophil-induced mammary epithelial cell damage was investigated using an in vitro model of co-culturing bovine neutrophils and MAC-T cells, a mammary epithelial cell line . Epithelial cell damages were evaluated by measuring lactate dehydrogenase activity in culture media and by morphological observations of cells after acridine orange staining . Activation of neutrophils with Escherichia coli lipopolysaccharide and phorbol 12-myristate 13-acetate caused superoxide and gelatinase release in media . Activated neutrophils damaged the epithelial cells, as demonstrated by an increase in lactate dehydrogenase release and the observation of morphological changes . The addition of melatonin or catalase reduced neutrophil-induced cytotoxicity in a dose-dependent manner, whereas superoxide dismutase and aprotinin had no effect on cytotoxicity . Melatonin has been reported to scavenge hydroxyl radical and peroxynitrite, whereas catalase and superoxide dismutase scavenge hydrogen peroxide and superoxide, respectively . Our results suggest that hydroxyl radicals released by activated bovine neutrophils cause damage to mammary epithelial cells and that antioxidants may be useful to protect the mammary tissue during bovine mastitis.

J Neurooncol, 2002 Jan, 56(1), 29 - 34
Interleukin-6-producing cells in a human glioblastoma cell line are not affected by ionizing radiation; Dubost JJ et al.; We investigated the production of interleukin 6 (IL-6) by a radioresistant human glioblastoma cell line G5 after single radiation events of 3, 6 and 9 Gy . The total cell number and IL-6 concentration in culture supernatant were assessed 24-96 h after irradiation . The radiation impeded or stopped G5 cell growth in a dose-dependent manner, but unexpectedly did not affect the IL-6 concentration in cell culture media that increased in the same range as in non-irradiated cultures . Furthermore, using flow cytometry, we found that the IL-6 positive cells expansion was unaffected by radiation . These findings suggested that this small (about 1%) fraction of G5 cells, constitutively producing IL-6, is highly radioresistant.

Resuscitation, 2002 Apr, 53(1), 93 - 9
Moderate hypothermia alters interleukin-6 and interleukin-1alpha reactions in ischemic brain in mice; Yanagawa Y et al.; Female C57BL/6 mice were decapitated and their brains were removed and cultured at either 37 or 33 degrees C for 48 h to investigate whether or not moderate hypothermia alters the cytokine reactions in the ischemic brain . The interleukin-6 and interleukin-1alpha levels in the culture media were significantly elevated in a time-dependent manner . The interleukin-6 levels after the incubation at 33 degrees C were significantly lower than those at 37 degrees C . The interleukin-1alpha levels at 33 degrees C were significantly higher than those at 37 degrees C . The interleukin-1alpha levels incubated with interleukin-6 antibody were significantly higher than those without IL-6 antibody . At 37 degrees C, the mRNA expression of interleukin-6 was observed from 2 to 48 h after incubation, but the same expression of interleukin-1alpha was only detected until 12 h . Accordingly, the ischemic brain incubated at 33 degrees C showed a decreased interleukin-6 production in comparison with that at 37 degrees C and the level of interleukin-6 showed negative feedback for the production of interleukin-1alpha . The temperature should, therefore, be carefully considered when evaluating the cytokine reaction for cerebral ischemia.

Atherosclerosis, 2002 May, 162(1), 131 - 5
Effect of HDL, from Japanese white rabbit administered a new cholesteryl ester transfer protein inhibitor JTT-705, on cholesteryl ester accumulation induced by acetylated low density lipoprotein in J774 macrophage; Kobayashi J et al.; We have previously reported a potent and specific cholesteryl ester transfer protein (CETP) inhibitor JTT-705 was a potentially anti-atherogenic compound (Nature 406 (2000) 203) . In the present study, we investigated in vitro how this compound affects properties of high density lipoprotein (HDL) in Japanese white (JW) rabbits in terms of reverse cholesterol transport in J774 macrophages . Plasma HDL-cholesterol (C) level was significantly higher in the rabbits administered JTT-705 than in control rabbits on days 3 and 7 . Both HDL(2) and HDL(3)-C levels were also significantly higher in JTT-705-administered rabbits than in control rabbits . During this period, plasma CETP activity was kept lower in JTT-705-administered rabbits than in controls . To determine how this compound affects the property of HDL particles, we investigated the C efflux induced by HDL from JTT-705-administered and control rabbits in J774 macrophages . Cholesterol ester (CE) concentration in J774 macrophages was reduced in proportion with increasing concentration of the added HDL to the culture media for J774 macrophages in both groups, suggesting that the HDL from JTT-705-administered rabbits was able to reduce CE concentration in J774 macrophages as efficiently as that from control rabbits . This result, together with the finding that the absolute HDL concentration increased in JW rabbits administered this CETP inhibitor, suggests that treatment with this new compound causes a beneficial effect on lipid metabolism in terms of anti-atherogenicity.

Cloning Stem Cells, 2001, 3(3), 115 - 23
Glucose and pyruvate metabolism of preimplantation goat blastocysts following in vitro fertilization and parthenogenetic activation; Ongeri EM et al.; The energy metabolism of preimplantation embryos can be used to predict viability and postimplantation development . Although preimplantation development and mean blastocyst cell numbers of goat in vitro-fertilized (IVF) embryos and chemically activated parthenogenotes are comparable, mammalian parthenogenotes are not viable, with most dying shortly after implantation . The objective of this study was to compare glucose and pyruvate metabolism of IVF goat blastocysts with that of parthenogenetic blastocysts developing from chemically activated oocytes . Embryos derived from IVF and parthenogenotes produced by exposing oocytes to either ionomycin or ethanol followed by 6-dimethylaminopurine (6-DMAP) were cultured in G1.2/G2.2 sequential culture media . Metabolism was determined for individual blastocysts using {5-3H}glucose and {2-14C}pyruvate to determine glycolytic and Kreb's cycle activity, respectively . Data were analyzed by ANOVA . A significantly higher percentage of activated oocytes underwent cleavage and developed to the blastocyst stage compared to IVF oocytes (p < 0.05) . There was no significant difference in glucose or pyruvate metabolism between IVF and parthenogenetically activated blastocysts . Mean glucose metabolism through glycolysis was 154.9 +/- 29.1, 130.3 +/- 17.1, and 129 +/- 16.5 pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively . Mean pyruvate metabolism through the Kreb's cycle was 28.1 +/- 8.0, 15.8 +/- 4.2, and 24.4 +/- 4.4 in pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively . Our results suggest that known differences in postimplantation development observed in IVF versus parthenogenetic embryos cannot be attributed to differences in pyruvate or glucose metabolism in the preimplantation blastocysts . Thus, these activation protocols result in embryos capable of appropriate regulation of key metabolic enzymes.

Biochem J, 2002 Jul 1, 365(Pt 1), 229 - 37
Mechanism of negative regulation of rat glutathione S-transferase A2 by the cytokine interleukin 6; Voss SH et al.; A decrease in concentration of some liver proteins, including the detoxification enzyme glutathione S-transferase A2 (rGSTA2), occurs during the acute-phase response . Interleukin 6 (IL-6) with dexamethasone (DEX) decreases transcription of rGSTA2 in rat hepatocytes . The promoter region that mediates suppression of rGSTA2 was localized to 150 bp . These 150 bp were divided and used for electrophoretic mobility-shift assays . Induction of a protein that specifically bound to an oligonucleotide from this region required new protein synthesis and IL-6 with DEX in the culture media . The protein bound to part of the hepatocyte nuclear factor 1 (HNF1) site but was different from and did not displace HNF1 . A core sequence, TGATT, was required for binding . The protein also bound to an HNF1 site in the albumin promoter . We hypothesize that IL-6 along with DEX induced a novel protein that decreased transcription of rGSTA2 and possibly albumin by interfering with the transactivating function of HNF1 . The protein may be an important negative regulator of transcription during the acute-phase response.

Microbiol Immunol, 2002, 46(2), 83 - 8
Development of a new medium useful for the recovery of dermatophytes from clinical specimens by minimizing the carryover effect of antifungal agents; Nakashima T et al.; Two surface-active compounds, egg lecithin and polysorbate 80, usually used as the deactivators of various preservatives were tested whether they also counteract either or all of the three major topical antifungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF) . Both egg lecithin and polysorbate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized the anti-dermatophytic activity of the three drugs in a concentration-dependent manner . A greater extent of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were added; MIC's of BFZ were increased more than 30-fold and those of LCZ and TBF more than 200-fold compared with the values obtained in the absence of the deactivators . Using the agar medium supplemented with the combined deactivators, culture studies were carried out with skin tissues specimens taken from guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical preparations of the three antifungal drugs . The experimental data from this animal study demonstrated that the combined deactivators-supplemented medium yielded increased numbers of fungi compared with the basal medium . It looks, therefore, likely that the fungal recovery on the former medium more correctly reflects to actual fungal burden in the infected lesions than the latter . All these results suggest that the combined deactivators-supplemented medium is more useful for mycological evaluation of therapeutic efficacy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.

Wei Sheng Yan Jiu, 1999 Mar 30, 28(2), 97 - 100
{Effect of antioxidant vitamins on lipid peroxide injury of aortic endothelial cells induced by oxidatively modified low-density lipoprotein in vitro}; He H et al.; The effect of antioxidant vitamins including vitamin E, vitamin C and beta-carotene on lipid peroxidation injury of aortic endothelial cells induced by oxidatively modified low density lipoprotein (oxLDL) were observed . Bovine aortic endothelial cells were incubated in culture media with antioxidant vitamins for 12 hours, and then with oxLDL (0.1 g/L) for 24 hours respectively . The results showed that the malondialdehyde (MDA) content in culture media of the groups with oxLDL was significantly higher than those of groups with vitamin E, vitamin C and beta-carotene alone . The number of HL60 monocyte cells adhered to bovine aortic endothelial cells was obviously higher in oxLDL groups than those in groups with only antioxidant vitamins . This study indicated that antioxidant vitamins are protective on vascular from injury induced by oxLDL, including decreasing lipid peroxidation and reducing adherence of monocyte cells on endothelia and seemed to be promising in the prevention of atherosclerosis.

Wei Sheng Yan Jiu, 1999 Mar 30, 28(2), 74 - 6
{The toxicity of combination of selenium, fluoride and arsenic on rat embryos}; Li Y et al.; Whole embryo rotated culture technique was used to investigate the toxicity of combination of selenium, fluoride and arsenic on rat embryos at day 9.5 of gestation . The result of factorial analysis (3 x 3 x 3) showed that the main effect of combination of selenium, fluoride and arsenic on the developmental toxicity was synergistic . The mixtures with different level of these three chemicals in combination could result in different developmental toxicity . The low level combinations mainly caused teratogenic effect, and the high level combinations(selenium 2.0 micrograms + fluoride 10 micrograms + arsenic 1.0 microgram/ml culture media) caused lethal effect . The results suggested that the disorders of yolk-sac placenta in structure and function were one of teratogenic mechanisms for the combination of selenium, fluoride and arsenic.

Biol Chem, 2002 Jan, 383(1), 167 - 76
The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures; Nezi L et al.; Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence . In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface . However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts . FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils . Incubation with plasmin inhibitors greatly reduces this effect . FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment . This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells . A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence . These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.

J Endocrinol, 2002 Apr, 173(1), 199 - 209
Effects of dibutyryl cAMP on stanniocalcin and stanniocalcin-related protein mRNA expression in neuroblastoma cells; Wong CK et al.; Stanniocalcin is a polypeptide hormone that was first reported in fish as a regulator of mineral metabolism . Its recent identification in mammals has opened a new area of investigation in basic and clinical endocrinology . In the present study, regulation of the stanniocalcin (STC) and stanniocalcin related protein (STCrP) genes were investigated in mouse neuroblastoma cells (Neuro-2A) in relation to neuronal cell differentiation . Neuro-2A is an undifferentiated cell line that contains measurable levels of STCrP mRNA, but undetectable levels of STC mRNA . Treatment of the cells with either dbcAMP (1-4 mM) or 50 microM euxanthone (PW1) resulted in extensive differentiation and neurite outgrowth . However, only neurites of dbcAMP-treated cells developed varicosities, a phenotypic marker of axon formation . Furthermore, following differentiation induced by dbcAMP, there was an upregulation of STC and downregulation of STCrP mRNA levels . In the first 24 and 48 h of treatments, there was a maximum twofold induction and 1.5-fold reduction in STC and STCrP mRNAs respectively . Following 96 h of treatment, an additional 14-fold STC induction and 1.2-fold STCrP reduction were observed . The increase in STC mRNA levels was accompanied by a concomitant increase in axon-specific low molecular form microtubule-associated protein (MAP-2c) mRNA and varicosities on the neurites, suggesting a possible role for STC in axonogenesis . There was no induction of STC mRNA levels when PW1 was added into the culture media, whereas ionomycin (1-10 microM) had no observable effects on cell differentiation or STC/STCrP mRNA . Immunocytochemical staining of dbcAMP-treated cells revealed abundant levels of immunoreactive STC, particularly in the varicosities, with only weak staining in control, untreated cells . Antisense oligodeoxynucleotides transfection studies indicated that the expression of STC was a cause of varicosity formation and a consequence of cell differentiation . Our findings lend further support to the notion that STC is involved in the process of neural differentiation.

J Nutr, 2002 Apr, 132(4), 674 - 9
Selenite and selenomethionine promote HL-60 cell cycle progression; Zeng H; The essential role of selenium (Se) in nutrition is well established . The elucidation of the mechanisms by which selenium regulates the cell cycle can lead to a better understanding of the nature of selenium's essentiality and its role in disease prevention . In this study, the effects of selenium deficiency or adequacy (0.25 micromol/L selenite or selenomethionine) on HL-60 cell cycle progression were examined in serum-free media . Selenium was critical for promotion of HL-60 cell growth . Cell-cycle analysis revealed that selenium deficiency caused a decrease in G1 phase cells that corresponded to an increase in G2 and sub-G1 phase cells . Gene array analysis suggested that c-Myc, cyclin C, proliferating cell nuclear antigen, cyclin-dependent kinase (cdk)1, cdk2, cdk4, cyclin B and cyclin D2 mRNA levels were lower in selenium-deficient cells than in the cells supplemented with 0.25 micromol/L selenomethionine . The decrease in the c-Myc mRNA level in selenium-deficient cells was confirmed by reverse transcription-polymerase chain reaction analysis . Furthermore, the phosphorylation state of total cellular protein was higher (57%) in selenium-supplemented cells than in selenium-deficient cells . Collectively, these results suggest a novel role for selenium at 0.25 micromol/L in up-regulation of the expression of numerous cell cycle-related genes and total cellular phosphorylated proteins in HL-60 cells in serum-free culture media . This leads to the promotion of cell cycle progression, particularly G2/M transition and/or the reduction of apoptosis, primarily in G1 cells . These observations may have additional implications for understanding the nature of selenium's essentiality.

J Biol Chem, 2002 Jun 7, 277(23), 20518 - 26 Epub 2002 Mar 29.
Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis; Englert RP et al.; Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms . In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl . The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated . When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2) . Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2) . In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis . HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg . The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.

Int J Legal Med, 2002 Feb, 116(1), 58 - 61
Preliminary approach to elucidate the role of pigment as a binding site for drugs and chemicals in anagen hair: differential uptake of 3H-haloperidol by pigment-producing compared to non-pigment-producing cell lines; Potsch L et al.; A striking difference was observed for cellular-bound drug in HaCaT and Sk-Mel-1 cells for a fixed drug exposure time of 72 h and varying 3H-haloperidol concentrations in the culture media . Drug uptake was dependent on drug concentration and linearly correlated for both the non-pigment- and the pigment-producing cells which however was different in magnitude . In an additional investigation the time course of drug uptake during 3H-haloperidol exposure (400 pmol/ml; 28 days) revealed increasing drug concentrations in the Sk-Mel-1 population, whereas drug concentrations in the keratinocytes reached a plateau within a short time period . In contrast to the HaCaT cells no tendency to saturation was observed for the pigment-producing cell line . At the end of the experiments 3H-haloperidol concentrations in Sk-Mel-1 were found to be approximately tenfold higher than in HaCaT.

Exp Neurol, 2002 Apr, 174(2), 230 - 42
Astrocyte delivery of glial cell line-derived neurotrophic factor in a mouse model of Parkinson's disease; Cunningham LA et al.; Primary astrocytes were genetically modified ex vivo to express recombinant glial cell line-derived neurotrophic factor (GDNF) and subsequently were tested for their ability to provide neuroprotection to dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease . A replication-defective retrovirus was constructed, which contained the rat GDNF sequence and a sequence encoding a beta-galactosidase (beta-gal)/neomycin phosphotransferase fusion protein, linked via an internal ribosomal entry site . Murine astrocytes transduced with this vector secreted GDNF into the culture media at the rate of 115 +/- 34 pg/24 h/10(5) cells and expressed cytoplasmic beta-gal, whereas control nontransduced astrocytes were negative for GDNF production and cytoplasmic beta-gal expression . Mice that received implants of GDNF-producing astrocytes into the striatum or nigra displayed elevated levels of GDNF compared to mice that received control nontransduced astrocytes . In addition, tissue content of GDNF was increased bilaterally and in brain regions both proximal and distal to the graft, even though astrocyte migration away from the graft site did not occur . Importantly, GDNF-producing astrocytes provided marked neuroprotection of nigral dopaminergic perikarya, and partial protection of striatal dopaminergic fibers, when implanted into the midbrain 6 days prior to a retrograde 6-OHDA lesion, as assessed by tyrosine hydroxylase immunohistochemistry . Similarly, GDNF-producing astrocytes prevented the acquisition of amphetamine-induced rotational behavior in 6-OHDA-treated mice and completely prevented dopamine depletion within the substantia nigra, as assessed by high-performance liquid chromatography . These results indicate that continuous exposure to low levels of GDNF provided by transgenic astrocytes provides marked neuroprotection of nigral dopaminergic neurons . (c)2002 Elsevier Science (USA).

Biochim Biophys Acta, 2002 Jan 30, 1580(1), 31 - 9
Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line; Carubbi F et al.; This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells . Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity . Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release . In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined . At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l . At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release . At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01) . In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05) . Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone . At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis . We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.

Neurochem Int, 2002 Jul, 41(1), 37 - 42
TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells; Yoon HY et al.; Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein . The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities . Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence . TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells . Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.

J Invest Dermatol, 2002 Apr, 118(4), 658 - 63
Increased expression of endothelial cell adhesion molecules due to mediator release from human foreskin mast cells stimulated by autoantibodies in chronic urticaria sera; Lee KH et al.; Histamine-releasing antibodies that act against the epitope of the alpha chain of Fc(epsilon)RI (anti-Fc(epsilon)RI(alpha) antibody) that may affect pathogenesis in serum of patients with chronic urticaria . We assessed the capability of anti-Fc(epsilon)RI(alpha) antibody in sera from patients with chronic urticaria to release histamine and cytokines, and to induce the expression of endothelial cell adhesion molecules . We also assessed the release of inflammatory mediators from cultured foreskin mast cells, and expression of endothelial cell adhesion molecules on human dermal microvascular endothelial cells . Cells were pretreated with mast cell-conditioned media: culture media of mast cells treated with sera from chronic urticaria patients containing anti-Fc(epsilon)RI(alpha) antibody . Histamine release from human foreskin mast cells challenged with sera, increased after both 20 min and 16 h intervals . Leukotriene D4 release also increased at both 20 min and 16 h . Tumor necrosis factor-alpha increased significantly in foreskin mast cell culture challenged with sera of chronic urticaria patients . After the stimulation of human dermal microvascular endothelial cells with the conditioned media, the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin increased significantly . Treatment of the conditioned media with anti-tumor necrosis factor-alpha monoclonal antibody partially inhibited the expression of intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin . The data suggest that sera from patients with chronic urticaria containing anti-Fc(epsilon)RI(alpha) antibody release mediators and tumor necrosis factor-alpha by activating human foreskin mast cells . This release can play a pathogenic role in chronic urticaria by activating endothelial cells, in part due to the actions of tumor necrosis factor-alpha from mast cells.

J Invest Dermatol, 2002 Apr, 118(4), 565 - 72
Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes; Boyce ST et al.; Cultured skin substitutes have become useful as adjunctive treatments for excised, full-thickness burns, but no skin substitutes have the anatomy and physiology of native skin . Hypothetically, deficiencies of structure and function may result, in part, from nutritional deficiencies in culture media . To address this hypothesis, vitamin C was titrated at 0.0, 0.01, 0.1, and 1.0 mM in a cultured skin substitute model on filter inserts . Cultured skin substitute inserts were evaluated at 2 and 5 wk for viability by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion . Subsequently, cultured skin substitute grafts consisting of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates were incubated for 5 wk in media containing 0.0 mM or 0.1 mM vitamin C, and then grafted to athymic mice . Cultured skin substitutes (n = 3 per group) were evaluated in vitro at 2 wk of incubation for collagen IV, collagen VII, and laminin 5, and through 5 wk for epidermal barrier by surface electrical capacitance . Cultured skin substitutes were grafted to full-thickness wounds in athymic mice (n = 8 per group), evaluated for surface electrical capacitance through 6 wk, and scored for percentage original wound area through 8 wk and for HLA-ABC-positive wounds at 8 wk after grafting . The data show that incubation of cultured skin substitutes in medium containing vitamin C results in greater viability (higher BrdU and MTT), more complete basement membrane development at 2 wk, and better epidermal barrier (lower surface electrical capacitance) at 5 wk in vitro . After grafting, cultured skin substitutes with vitamin C developed functional epidermal barrier earlier, had less wound contraction, and had more HLA-positive wounds at 8 wk than without vitamin C . These results suggest that incubation of cultured skin substitutes in medium containing vitamin C extends cellular viability, promotes formation of epidermal barrier in vitro, and promotes engraftment . Improved anatomy and physiology of cultured skin substitutes that result from nutritional factors in culture media may be expected to improve efficacy in treatment of full-thickness skin wounds.

Med Hypotheses, 2001 Dec, 57(6), 697 - 700
Stem cell strategies for neuroreplacement therapy in Alzheimer's disease; Sugaya K et al.; The existence of neural stem cells (NSCs) in the adult human brain provides impetus for investigating possible neuroreplacement therapies for neurodegenerative disease . Due to recent advances in techniques affording isolation and maintenance of NSCs using non-serum culture media, these cells have become exciting candidates for therapeutic strategies . We are able to expand NSCs by mitogenic growth factors in vitro and in defined conditions, NSCs differentiate into each of the diverse brain cell types: neurons, astrocytes and oligodendrocytes . This article addresses the involvement of amyloid-beta precursor protein and the presenilins in NSCs' biology and possible application of NSCs for therapeutic approaches in Alzheimer's disease . Ongoing studies in our laboratory, and recent findings by others using human neural progenitors, serve as the conceptual frame for this article.

Eur J Cancer Prev, 2001 Dec, 10(6), 507 - 13
In vitro and in vivo (SCID mice) effects of phytosterols on the growth and dissemination of human prostate cancer PC-3 cells; Awad AB et al.; The dietary effect of phytosterols (PS) versus cholesterol on the growth and metastasis of the PC-3 human prostate cancer cells in SCID mice was studied . Also, their direct effect on the growth and migration of these cells in vitro was analysed . In the in vivo experiment, SCID mice were fed a diet containing 2% of either PS mixture or cholesterol plus 0.2% cholic acid and implanted with 2 x 10(6) tumour cells per mouse . Tumour growth was monitored for 8 weeks post inoculation . Animals fed the PS diet had tumours 40-43% smaller than those fed the cholesterol diet . Furthermore, the number of mice with lymph node and lung metastasis was almost one-half that of the cholesterol-fed group . In the in vitro studies, both beta-sitosterol and campesterol inhibited the growth of PC-3 cells by 70% and 14%, respectively, while cholesterol supplementation increased the growth by 18% when compared with controls . PS inhibited the invasion of PC-3 cells into Matrigel-coated membranes by 78% while cholesterol increased it by 43% as compared with the cells in the control media . Migration of tumour cells through 8 microm pore membranes was reduced by 60-93% when the PC-3 cells were in PS media, as compared with a 67% increase after cholesterol supplementation . PS supplementation reduced the binding of PC-3 cells to laminin by 15-38% and fibronectin by 23% while cholesterol increased binding to type IV collagen by 36% . It was concluded that PS indirectly (in vivo as a dietary supplement) and directly (in tissue culture media) inhibited the growth and metastasis of PC-3 cells . beta-Sitosterol was more effective than campesterol in offering this protection in most of the parameters studied.

Br J Ophthalmol, 2002 Apr, 86(4), 412 - 7
Systemic inflammation and innate immune response in patients with previous anterior uveitis; Huhtinen M et al.; AIM: To determine the presence of systemic inflammation and innate immune responsiveness of patients with a history of acute anterior uveitis but no signs of ocular inflammation at the time of recruitment . METHODS: Tumour necrosis factor alpha (TNF-alpha) production in response to bacterial lipopolysaccharide (LPS) was studied using whole blood culture assay; levels of TNF-alpha in culture supernatants, and soluble interleukin 2 receptor (sIL-2R) in serum were determined by chemiluminescent immunoassay (Immulite); monocyte surface expression of CD11b, CD14, and CD16 and the proportion of monocyte subsets CD14(bright)CD16(-) and CD14(dim)CD16(+) were studied with three colour whole blood flow cytometry; and serum C reactive protein (CRP) levels were determined using immunonephelometric high sensitivity CRP assay . RESULTS: The CRP level (median, interquartile range) was significantly higher in 56 patients with previous uveitis than in 37 controls (1.59 (0.63 to 3.47) microg/ml v 0.81 (0.32 to 2.09) microg/ml; p=0.008) . The TNF-alpha concentration of the culture media per 10(5) monocytes was significantly higher in the patient group than in the control group in the presence of LPS 10 ng/ml (1473 (1193 to 2024) pg/ml v 1320 (935 to 1555) pg/ml; p=0.012) and LPS 1000 ng/ml (3280 (2709 to 4418) pg/ml v 2910 (2313 to 3358) pg/ml; p=0.011) . The background TNF-alpha release into the culture media was low in both groups . CD14 expression of CD14(bright)CD16(-) monocytes, defined as antibody binding capacity (ABC), was similar for the patients and controls (22,839 (21,038 to 26,020) ABC v 21,657 (19,854 to 25,646) ABC) . CONCLUSIONS: Patients with previous acute anterior uveitis show high innate immune responsiveness that may play a part in the development of ocular inflammation.

Biol Reprod, 2002 Apr, 66(4), 1178 - 84
Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos; Chung YG et al.; Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo . Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined . To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei . We find that glucose is beneficial during the 1-cell stage when CZB medium is employed . We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media . Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM . Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone . The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst . Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos . The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.

Reproduction, 2001 Dec, 122(6), 865 - 73
Effect of nitric oxide on the expression of insulin-like growth factors and the insulin-like growth factor binding proteins throughout the lifespan of the human corpus luteum; Iniguez G et al.; The presence of insulin-like growth factors (IGF), IGF binding proteins (IGFBP) and IGF receptor type 1 (IGF-IR) in the human corpus luteum was investigated by examining the expression and production of related proteins throughout the lifespan of the corpus luteum and the action of nitric oxide upon their production . The expression of proteins in corpora lutea from the early, mid-and late luteal phases was assessed by immunohisto-chemistry, evaluated by a semi-quantitative analysis and the functional study was performed in corpus luteum explants incubated with nitric oxide donors . IGF-I and -II and IGFBP-1 and -3 were measured in the culture media by specific immunoassays . The results showed that IGF-I and -II, IGFBP-1 to -6 and IGF-IR were detected in the human corpus luteum throughout the luteal phase . Moreover, the expression and production of IGF-I and IGFBP-1 increased progressively from corpora lutea from the early to late luteal phases (P < 0.05), whereas the expression and production of IGFBP-2, -4 and -5 were significantly higher in corpora lutea from the mid-luteal phase (P < 0.05) . No differences were observed in the expression of IGF-II, IGFBP-3 and -6 and IGF-IR throughout the lifespan of the corpus luteum . However, functional studies showed that nitric oxide donors elicited a stimulatory action on production of IGF-I in corpora lutea from the early luteal phase (80%) and on production of IGFBP-1 in corpora lutea from the late luteal phase (50%) (P < 0.05), whereas production of IGF-II and IGFBP-3 was not affected by nitric oxide . In conclusion, the components of the IGF-IGFBP system are expressed in the human corpus luteum throughout its lifespan . Nitric oxide regulates IGF-I and IGFBP-1 production, indicating that the growth factors may serve, at least in part, as mediators of the action of nitric oxide in the human corpus luteum.

Cloning, 2001, 3(2), 41 - 50
Effect of culture media on in vitro development of cloned mouse embryos; Heindryckx B et al.; The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied . Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2 . Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities . Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique . Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%) . Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2 . Arrested two- and three-cell stage NT embryos showed a high rate of binucleation . These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.

J Periodontol, 2002 Feb, 73(2), 213 - 9
The effects of varying degrees of allograft decalcification on cultured porcine osteoclast cells; Herold RW et al.; BACKGROUND: Demineralized freeze-dried bone allograft (DFDBA) is widely used in periodontal therapy as a scaffold for new bone formation in periodontal defects . It is demineralized, theoretically, to expose osteoinductive or osteoconductive bone matrix proteins that should facilitate osteogenesis . The degree of DFDBA demineralization varies between tissue banks and may affect clinical regeneration . A 2% residual calcium level in DFDBA has been shown to result in the highest alkaline phosphatase activity levels in cultured human periosteal cells and is optimally osteoinductive or osteoconductive for new bone formation . The purpose of this study was to evaluate the effect of 4 different residual calcium levels in commercially available DFDBA samples on porcine osteoclast activity as measured by resorption on calcium phosphate-coated disks . METHODS: Bone marrow was harvested from the femurs of 3-week-old farm pigs and cultured for 3 weeks . Hematopoietic stem cells were allowed to differentiate into mature active polykaryons displaying genuine osteoclast characteristics . The osteoclast cells displayed a dense actin band inside the margins of the cytoplasm under light microscopy . Culture media was decanted and collagenase added to free the attached cells . Equal cell samples were pipetted onto calcium phosphate-coated disks in 24-well plates . DFDBA samples with 1.44%, 2.41%, and 5.29% residual calcium; FDBA (30% residual calcium); and control cultures without allograft samples were prepared and all samples incubated for 1 week . Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), Oregon Green 488-phalloidin, a stain for cytoskeletal proteins, and counterstained with propidium iodide . Specimens were examined by light and fluorescence microscopy using epi-illumination . Calcium phosphate disks were then rinsed in 5% sodium hypochlorite to remove adherent osteoclasts, and substrate surface changes were measured by white light interferometry and image analysis . RESULTS: A higher yield of TRAP-positive cells was produced without DFDBA; however, resorptive activity appears to be significantly increased in the presence of 2.41% residual calcium as compared to all other experimental groups (P<0.0065) . CONCLUSION: In this in vitro model, porcine osteoclasts show significantly more resorptive activity as measured on calcium phosphate-coated disks in the presence of 2.41% residual calcium in DFDBA than in other DFDBA residual calcium levels.

J Magn Reson Imaging, 2002 Mar, 15(3), 315 - 23
Effect of IL-1beta-induced macromolecular depletion on residual quadrupolar interaction in articular cartilage; Borthakur A et al.; PURPOSE: Sodium multiple-quantum filtered (MQF) NMR spectroscopy may potentially be used to measure proteoglycan (PG) depletion in cartilage caused by osteoarthritis (OA) . The purpose of this work was to quantify the effect of interleukin-1 (IL-1beta)-induced macromolecule depletion on the residual quadrupolar interaction (RQI) of sodium in bovine cartilage plugs . MATERIALS AND METHODS: Fifteen 8-mm-diameter cartilage plug specimens were cored from the articular surface of fresh bovine patellae . All plugs were kept in culture media and nine of the plugs were subjected to interleukin-1 (IL-1beta)-induced degeneration of cartilage for 4, 6, and 7 days . Sodium NMR spectra were obtained from each sample with a 1-cm-diameter solenoid coil in a 2T whole-body magnet interfaced to a custom-built spectrometer . We employed a previously described theoretical model to analyze triple-quantum filtered (TQF) and double-quantum filtered magic angle (DQFMA) spectra obtained from normal cartilage and cartilage treated with IL-1beta . The model assumes a static Gaussian distribution of the RQI frequency, omega(Q), in the sample . TQF and DQFMA spectra from each sample were fitted with the appropriate signal expressions to determine sigma (the root mean square (RMS) omegaQ), T2f, and T2s . An inversion-recovery sequence was used to determine T1 of each plug . A spectrophotometric assay was used to determine the amount of PG depleted from each plug . Histology was performed to visualize the PG loss in cartilage plugs . We defined sigma as the measure of changes in macroscopic order in the tissue . RESULTS: Simulated spectra from the theoretical model were in excellent agreement with the experimental data . We were able to determine the relaxation times as well as sigma of each specimen from their corresponding fits . T2f ranged between 2.26-3.50 msec, decreasing with increased PG loss . Over the range of PG depletion investigated, T2s increased from 12.3 msec to 14.9 msec, and T1 increased from 16 msec to 21 msec, while sigma decreased from 180 Hz to 120 Hz . The order of macromolecules in the cartilage tissue decreased substantially with PG loss . Histology sections clearly showed qualitative visualization of the PG loss in cartilage following treatment with IL-1beta . CONCLUSION: We demonstrated that IL-beta-induced macromolecule depletion in cartilage not only changes the relaxation characteristics of sodium but also changes RQI of the tissue . Using MQF sodium spectroscopy we quantified the changes in sigma and showed that loss of macromolecules reduces the degree of order in the tissue.

J Comp Neurol, 2002 Apr 1, 445(2), 176 - 98
Hair cell development in vivo and in vitro: analysis by using a monoclonal antibody specific to hair cells in the chick inner ear; Kondo K et al.; The purpose of this study was to establish a hair cell-specific marker and a convenient explant culture system for developing chick otocysts to facilitate in vivo and in vitro studies focusing on hair cell genesis in the inner ear . To achieve this, a hair cell-specific monoclonal antibody, 2A7, was generated by immunizing chick inner ear tissues to a mouse . Through the use of immunofluorescence and immunoelectron microscopy, it was shown that 2A7 immunoreactivity (2A7-IR) was primarily restricted to the apical region of inner ear hair cells, including stereocilia, kinocilia, apical membrane amongst the extending cilia, and superficial layer of the cuticular plate . Although the 2A7 antibody immunolabeled basically all of the hair cells in the posthatch chick inner ear, two different patterns of 2A7-IR were observed; hair cells located in the striolar region of the utricular macula, which consist of two distinct cell types identifiable on the basis of the type of nerve ending, Type I and II hair cells, showed labeling restricted to the basal end of the hair bundles . On the other hand, hair cells in the extrastriolar region, which are exclusively of Type II, showed labeling extending over virtually the entire length of the bundles . These findings raised the possibility that chick vestibular Type II hair cells, characterized by their bouton-type afferent nerve endings, can be divided into two subpopulations . Analysis of developing inner ear by using the 2A7 antibody revealed that this antibody also recognizes newly differentiated immature hair cells . Thus, the 2A7 antibody is able to recognize both immature and mature hair cells in vivo . The developmental potential of embryonic otocysts in vitro was then assessed by using explant cultures as a model . In this study, conventional otocyst explant cultures were modified by placing the tissues on floating polycarbonate filters on culture media, thereby allowing the easy manipulation of explants . In these cultures, 2A7-positive hair cells were differentiated from dividing precursor cells in vitro on the same schedule as in vivo . Furthermore, it was found that hair cells with both types of 2A7-IR were generated in culture as in vivo, indicating that a maturational process of hair cells also occurred . All these results as presented here suggest that the 2A7 monoclonal antibody as a hair cell-specific marker together with the culture system could be a potential tool in analysis of mechanisms underlying hair cell development .

Perit Dial Int, 2001, 21 Suppl 3, S35 - 40
High glucose dialysis solutions increase synthesis of vascular endothelial growth factors by peritoneal vascular endothelial cells; Seo MJ et al.; OBJECTIVE: Increased peritoneal vasculature has been reported in long-term peritoneal dialysis (PD), and vascular endothelial growth factors (VEGFs) have been found in dialysate . High concentrations of glucose or lactate, glucose degradation products (GDPs), and low pH of dialysis solutions are all possible factors in increased peritoneal VEGF synthesis . In this study, we investigated the effects of high glucose dialysis solutions on VEGF synthesis by peritoneal vascular endothelial cells (PVECs) . METHODS: The PVECs were isolated from rat omentum and were incubated for 4 hours in three different culture media {M199 media (control), conventional dialysis solutions containing 4.25% glucose diluted with an equal volume of M199 media (HGD), and M199 media containing 118 mmol/L mannitol as an osmolar control (mannitol)} . Levels of VEGF protein in the culture supernatant were measured by ELISA, and mRNA expression was determined by Northern blot analysis . Data are presented as percent of control . RESULTS: After incubation for 4 hours, the number of cells did not differ between the 3 groups . Levels of VEGF in culture supernatant were significantly higher in the HGD group (124% +/- 19%, p = 0.006) as compared with the control and mannitol (85% +/- 10%) groups.The mRNA expression of VEGF appeared to be higher in the HGD group (128% +/- 49%) than in the control and mannitol (94% +/- 18%) groups . CONCLUSION: High glucose dialysis solutions increased VEGF synthesis by PVECs . The relationship between VEGF synthesis by PVECs and neovascularization of the peritoneum observed in long-term peritoneal dialysis patients has to be studied further.

Gac Med Mex, 2002 Jan-Feb, 138(1), 1 - 13
{Morphologic and electrophysiologic characteristics of cultured vestibular ganglia neurons}; Soto E et al.; Vestibular afferent neurons have been classified on the basis of their spontaneous activity as regular and irregular; this has been attributed to their synaptic input, but it remains to be defined the participation of some intrinsical properties of the afferent neurons in the determination of their discharge pattern . In this work, we have developed tissue cultures of the rat vestibular ganglia . Isolated cells were plated using poly-D-lysine or collagen as substrates and L-15 or Neurobasal as culture media . After 48 hrs cells in the four experimental conditions give forth neurites of variable longitude . By using antibodies against the neurofilaments 160 kDa the cell structure was studied . Monopolar (30.6%), bipolar (63.9%) and multipolar (5.5%) cells were found . By using the voltage and current clamp procedures the voltage dependence and kinetics of the tetrodotoxin sensitive Na+ current was fully characterized . Cultured cells were shown to generate action potentials under electrical stimulation, and they were capable of repetitive spike discharge under the influence of 4-aminopyridine . These results demonstrate that tissue cultures constitute an excellent system to study the intrinsical properties of vestibular afferent neurons.

Spine, 2002 Mar 15, 27(6), 576 - 80
Effect of chondroitinase ABC on matrix metalloproteinases and inflammatory mediators produced by intervertebral disc of rabbit in vitro; Sakuma M et al.; STUDY DESIGN: Lumbar intervertebral discs in rabbit were cultured in the presence of chondroitinase ABC . The matrix metalloproteinases (MMPs) and inflammatory mediators produced in culture media were then analyzed . OBJECTIVES: To investigate the effect of chondroitinase ABC on MMPs and inflammatory mediators produced by intervertebral disc of rabbit in vitro . SUMMARY OF BACKGROUND DATA: The chemonucleolytic effect of chondroitinase ABC is caused by the decrease in the chondroitin sulfate, hyaluronan, and protein content of the nucleus pulposus in rabbit . The reason for the decreases in protein content remains unclear . METHODS: Anulus fibrosus and nucleus pulposus were cultured for 72 hours with or without chondroitinase ABC stimulated or not stimulated by interleukin-1 after preculture for 4 days . Subsequently, the MMPs (gelatinases MMP-2, MMP-9, and collagenase) and inflammatory mediators (prostaglandin E2 and nitric oxide) produced in the culture media were analyzed . RESULTS: In the anulus fibrosus chondroitinase ABC and interleukin-1 synergistically increased the collagenase activity, which was at a significantly higher level than the increment solely due to interleukin-1 . In contrast, chondroitinase ABC counteracted the increase in nitric oxide production by interleukin-1 . In the nucleus pulposus the collagenase and nitric oxide productions were not particularly affected by chondroitinase ABC and/or interleukin-1 . In zymographic analysis MMP-2 was detected, but MMP-9 was only slightly detected in both tissues . There were no significant differences in both tissues for MMP-2 and prostaglandin E2 following incubation with or without chondroitinase ABC, whether stimulated by interleukin-1 or not . CONCLUSIONS: The collagenase activity in the anulus fibrosus was increased by chondroitinase ABC with interleukin-1 . This finding may support the hypothesis that some proteolytic activities are involved in the chemonucleolytic process by chondroitinase ABC treatment.

JOP, 2002 Jan, 3(1), 8 - 15
Analysis and optimization of nutritional set-up for murine pancreatic acinar cells; Kurup S et al.; CONTEXT: Pancreatic acinar cell cultivation poses a serious problem due to limitations in the in vitro survival time despite variations of dissociation protocols, culture media and nutrient supplements . OBJECTIVE: To establish a long term culture of murine pancreatic acinar cells which retain their viability, monolayer formation and responsiveness to secretagogues . In order to investigate the mechanism of the short-life of acinar cells studied in vitro, we studied their survival under the influence of different supplements on nutrient media . INTERVENTIONS: Dissociated pancreatic acini were prepared from BALB/c mice pancreata by collagenase digestion supplemented with bovine serum albumin fraction V and soybean trypsin inhibitor . A nutrient set-up was designed for their long term survival in vitro . RESULTS: It was observed that mouse pancreatic acinar cells dissociated in presence of bovine serum albumin fraction V and soybean trypsin inhibitor result in 95% viability . Further cultivation of these acinar cells in Waymouth's MB 752/1 medium supplemented with 10% fetal calf serum (v/v), soybean trypsin inhibitor, bovine serum albumin, dexamethasone, and epidermal growth factor results in their survival for more than 6 days in culture with 85% viability, retention of the secretagogue responsiveness and formation of a monolayer without any extracellular matrix coating . CONCLUSIONS: Our study clearly demonstrates that the addition of soybean trypsin inhibitor to culture medium reduces zymogen granule fragility and acinar cell death, thus increasing their viability for sufficiently long periods . The present study offers an excellent, in vitro model for the investigation of exocrine dysfunction in response to acinar cell injury.

Toxicol Appl Pharmacol, 2002 Feb 15, 179(1), 13 - 20
Cadmium- and mercury-induced intercellular adhesion molecule-1 expression in immortalized proximal tubule cells: evidence for a role of decreased transforming growth factor-beta1; Jiang J et al.; A definitive association between the aberrant expression of cytokines and adhesion molecules in renal failure has been established . In this regard a relationship between cytokine and adhesion molecule expression is suggested but has not been shown in models of proximal tubular cell injury . To investigate the impact of acute injury on the relationship between transforming growth factor-beta 1 (TGF-beta1) and intercellular adhesion molecule-1 (ICAM-1) expression, two immortalized mouse proximal tubular epithelial cell lines were exposed to cadmium chloride or mercuric chloride (0-50 microM) for 0-8 h . ELISA and Western blot measured expression of secreted and intercellular TGF-beta1, respectively . Direct cellular ELISA or Western blot was used to assess ICAM-1 expression . Challenge with cadmium caused a greater loss of cell viability than did mercury . Interestingly, cadmium significantly decreased the amount of TGF-beta1 in the conditioned media . Although a similar trend was seen in mercury-challenged cells, no significant differences were observed . The decrease in TGF-beta1 in the culture media was not due to decreased expression of this cytokine, as intercellular levels were not affected by metal-induced injury . Significant increases in ICAM-1 protein expression were observed following cadmium and mercury challenge . The increase in ICAM-1 appears to be due to increased mRNA, as Northern blot analysis demonstrated increased message expression following a 4-h cadmium or mercury challenge . Supplementation of the culture media with exogenous TGF-beta1 decreased basal ICAM-1 expression and attenuated the cadmium-induced increase . These data suggest that metal-induced injury is associated with increased ICAM-1 expression . The mechanism of this induction may involve the decreased TGF-beta1 in the conditioned media following metal challenge . Taken together, these studies suggest a link between cytokine and adhesion molecule expression in renal injury.

Life Sci, 2002 Feb 8, 70(12), 1359 - 67
Trypsin is produced by and activates protease-activated receptor-2 in human cancer colon cells: evidence for new autocrine loop; Ducroc R et al.; In this work, we showed that human colon cancer cell lines produce trypsin which can activate a receptor for trypsin, the protease-activated receptor-2 (PAR-2), in these cells . RT-PCR experiments showed that trypsinogen transcripts were present in four colon cancer cell lines: T84, Caco-2, HT-29 and C1.19A . By Western blot analysis we found a 25 kDa immunoreactive band identified as trypsinogen I in cell lysates and in the corresponding culture media . Concentrations of trypsin in cell media were found in nanomolar range, thus compatible with activation of protease-activated receptor 2 (PAR-2) . This was further demonstrated in a colon cancer cell line (H-29) Ca2+i assay since increases in Ca2+i were observed in response to media from T84, Caco-2 or C1.19A cells that were similar to that observed with 2-5 nM trypsin and were abolished by trypsin inhibitor . Altogether, these data show that colon cancer cell lines produce and secrete trypsin at concentrations compatible with activation of PAR-2 . They support possible autocrine/paracrine regulation of PAR-2 activity by trypsin in colon cancer cells.

Pathol Oncol Res, 2001, 7(4), 251 - 9
Expression of thrombospondin-1 in human pancreatic adenocarcinomas: role in matrix metalloproteinase-9 production; Qian X et al.; Human pancreatic adenocarcinoma, an aggressive malignant disease, shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissues . Thrombospondin-1 (TSP-1), a 450 kDa platelet and matrix glycoprotein, has been implicated in tumor invasion, angiogenesis and metastasis . TSP-1 and MMP-9 expression in pancreatic adenocarcinoma and control pancreas tissues was measured by immunohistochemistry . TSP-1 expression in pancreatic carcinoma cell lines, fibroblasts, and endothelial cells was measured by a competitive TSP-1 enzyme linked immunosorbent assay (ELISA) . The effect of TSP-1 on MMP-9 production in pancreatic carcinoma cell lines was measured by zymography and Western blot analysis . Eighty five per cent (23/27) of cases of pancreatic adenocarcinoma showed increased TSP-1 staining in the desmoplastic stroma adjacent to tumor cells . No specific positive staining for TSP-1 was observed in the normal pancreatic tissues and the inflammatory areas . TSP-1 localized in tumor stroma surrounding the tumor cells expressing MMP-9 . Using TSP-1 competitive ELISA, the secretion of TSP-1 by different pancreatic cancer cell lines into culture medium varied from 11.45 plus minus 14.08 to 275.82 plus minus 45.56 ng/10 6 cells/24 hours . The amounts of TSP-1 detected in both culture media and cell extracts from fibroblasts or endothelial cells were at least 2-3 fold higher than those from pancreatic cancer cells . TSP-1 augmented the production of matrix metalloproteinase-9, a matrix degrading enzyme, in pancreatic cancer cells in vitro . Stromally-derived TSP-1 up-regulates the production of MMP-9 by pancreatic adenocarcinoma . These data are consistent with the conclusion that TSP-1-rich stroma is involved in regulating matrix remodeling in tumor invasion.

Microbiology, 2002 Mar, 148(Pt 3), 677 - 84
Influence of activated charcoal, porcine gastric mucin and beta-cyclodextrin on the morphology and growth of intestinal and gastric Helicobacter spp; Taneera J et al.; Bile-tolerant Helicobacter spp . are emerging human and animal pathogens . However, due to their fastidious nature, which requires nutrient-rich complex media to grow, infection with these bacteria may be underestimated . The accumulation of toxic metabolites in cultures may be one of the main obstacles for successful culture of these organisms . The present study examined various potential growth-enhancing substances for Helicobacter spp . and, furthermore, how they may affect spiral to coccoid conversion . Five Helicobacter spp . were cultured on agar and in broth media supplemented with activated charcoal, beta-cyclodextrin, or porcine gastric mucin . Growth was determined by estimating the numbers of colony-forming units and colony diameter, as well as bacterial cell mass . Coccoid transformation was estimated every 24 h by both Gram and acridine-orange staining . Activated charcoal was superior in supporting growth and increased cell mass on agar and in broth media . beta-Cyclodextrin delayed spiral to coccoid conversion by Helicobacter pylori and Helicobacter canis, whereas activated charcoal delayed the conversion to coccoid forms of Helicobacter hepaticus and Helicobacter bilis . The progression to coccoid forms by Helicobacter pullorum on agar media was not influenced by any growth supplement . The spiral to coccoid conversion was more rapid in broth media than on agar media . The growth enhancement observed is probably related to the capacity of activated charcoal to remove toxic compounds in culture media.

J Pediatr Surg, 2002 Mar, 37(3), 467 - 71
VEGF upregulates Bcl-2 expression and is associated with decreased apoptosis in neuroblastoma cells; Beierle EA et al.; BACKGROUND/PURPOSE: Both the expression of Bcl-2 and the amount of vascular endothelial growth factor (VEGF) are increased in neuroblastoma cells cocultured with hepatocytes . The authors hypothesize that VEGF upregulates Bcl-2 expression by the neuroblastoma cells and protects them from apoptotic stimuli . METHODS: To determine whether VEGF will induce Bcl-2 expression in neuroblastoma cells, the cells are plated with standard media (control) or media supplemented with VEGF . After 24 hours, Bcl-2 expression is measured . To determine whether VEGF protects neuroblastoma cells from apoptosis, the cells are subjected to tumor necrosis factor alpha (TNF-alpha) or serum starvation to induce apoptosis either with or without VEGF added to the culture media . The cells are collected and apoptosis measured using the deoxynucleotidyltransferase-mediated dUTP neck end labeling (TUNEL) method . RESULTS: VEGF increases Bcl-2 expression by 33% over cells cultured in standard media . Serum starving the tumor cells or adding TNF-alpha significantly increases the percentage of apoptotic cells . The addition of VEGF significantly protects the neuroblastoma cells from the apoptotic effects of both serum starvation and TNF-alpha . CONCLUSIONS: VEGF increases the expression of Bcl-2 and also abrogates TNF-alpha and serum starvation-induced apoptosis in neuroblastoma cells in vitro . VEGF may promote neuroblastoma survival not only through angiogenesis, but also by altering apoptosis and its regulating proteins .

Zhonghua Shao Shang Za Zhi, 2001 Aug, 17(4), 222 - 4
{Effects of tetrandrine on the synthesis of collagen and scar-derived fibroblast DNA}; Liu D et al.; OBJECTIVE: To investigate the effects of tetrandrine on the synthesis of collagen and scar derived fibroblast DNA . METHODS: Scar-derived human fibroblasts were cultured in vitro . The changes in the levels of the synthesis of collagen and DNA of the fibroblasts were represented by the incorporation values of (3)H-TdR and (3)H-proline into the cells . Tetrandrine was added to the culture media of the cells, and its effects were studied . RESULTS: When the concentration of added tetrandrine increased from 5 mg/L to 80 mg/L, The (3)H-TdR values in scar-derived fibroblasts were 1162 plus minus 226 and 412 plus minus 82, respectively while that in control group was 1740 plus minus 165, showing an inhibition rate of 76.32%, and the difference (P < 0.01) . In addition, the (3)H-proline incorporation values in the cells were 535 plus minus 141 and 341 plus minus 89, respectively, while that in control group was 1126 plus minus 193, with the inhibition rate of 69.71%, showing significant difference (P < 0.01) . CONCLUSION: The synthesis of DNA and collagen in cultured scar-derived fibroblasts could be inhibited by tetrandrine in dose-dependent pattern . Tetrandrine might be a potential agent for the prevention and treatment of proliferative scars.

Zhonghua Shao Shang Za Zhi, 2001 Feb, 17(1), 13 - 5
{The effects of DiBaiRen decoction on the proliferation of human keratinocytes and its in vitro anti--oxidation role}; Hu D et al.; OBJECTIVE: To investigate the effects of DiBaiRen decoction on the proliferation of human keratinocytes and its in vitro anti -- oxidation role . METHODS: Human keratinocytes were cultured in vitro in the media containing gradient diluted DiBaiRen decoction . The cell proliferation rates at different culture times were recorded . Cellular oxidation injury inflicted by superoxide anion and hydroxyl anion was produced by adding hypoxanthine -- xanthine oxidase and hydrogen peroxide to the culture media . MTT colorimetry was employed to determine cellular activity . The antagonistic effects of DiBaiRen decoction on cellular oxidation injury were analyzed . RESULTS: In the case that DiBaiRen decoction was added to the culture in concentrations of 30 ml/L to 100 ml/L, there exhibited obvious accelerated proliferation of the keratinocytes, with evident shortening of the cell fusion time . In addition, the oxidation injury of cultured keratinocytes caused by hypoxanthine -- xanthine oxidase and hydrogen peroxide was ameliorated or eliminated by the addition of DiBaiRen decoction, and the effects were dose -- dependent . CONCLUSION: DiBaiRen decoction might possess anti -- oxidation effects and could promote the in vitro proliferation of cultured human keratinocytes, and it might be beneficial in wound protection and epithelization.

J Viral Hepat, 2002 Mar, 9(2), 149 - 56
Full-length sequence and functional analysis of hepatitis B virus genome in a virus carrier: a case report suggesting the impact of pre-S and core promoter mutations on the progression of the disease; Kajiya Y et al.; In chronic hepatitis B virus (HBV) infection, the quiescent immunotolerant phase evolves into the immunoactive phase . The aim of the present study was to clarify the virological alterations relevant to progression . Serial serum samples obtained from a patient with HBV during long-term follow-up were analysed by sequencing of the full-length HBV-DNA using polymerase chain reaction (PCR) . In addition, PCR products of HBV genome from each serum sample were transfected into HuH-7 human hepatoma cells for the functional analysis of the transfected viral genomes . Based on the HBV-DNA sequence analysis, the patient had the genotype C virus, and the mutant HBV with common core promoter mutations (T(1762)A(1764)) and deletion of the pre-S region responsible for large surface protein transcription emerged before the onset of hepatitis . When the vigorous host immune response developed (indicated by the flare-up of hepatitis), the mutant HBV containing common core promoter mutations and another pre-S deletion causing lack of the surface protein promoter became predominant . The HBV-DNA sequences, other than pre-S and core promoter regions were identical to the wild-type sequence throughout the study . Transfection of PCR products containing the mutant HBV sequences resulted in increased amounts of intracellular replicative intermediates but the decreased secretion of HBsAg and HBeAg into culture media, suggesting accumulation of nonenveloped viral core particles within the cells . These results indicate that pre-S deletion and core promoter mutations may participate cooperatively in progression of the disease.

J Endocrinol, 2002 Mar, 172(3), 557 - 63
Biphasic regulation of activin A secretion by gonadotropins in cultured human ovarian granulosa-luteal cells leads to decreasing activin:inhibin ratios during continuing gonadotropin stimulation; Vanttinen T et al.; Pituitary gonadotropins mediate part of their effects on ovarian function via local hormones and growth factors produced by granulosa cells . Activins and inhibins are among these factors, and they have often opposite effects on various components of the reproductive system . The purpose of this study was to investigate the regulation of ovarian activin A secretion using cultured human ovarian granulosa-luteal cells as a model . The granulosa-luteal cells, obtained from women taking part in an in vitro fertilization program, were cultured and treated with FSH, LH, 8-bromo cAMP (8-BrcAMP, a protein kinase A activator) and 12-O-tetradecanoyl phorbol-13-acetate (TPA, a protein kinase C activator) . Conditioned cell culture media were analyzed for activin A, inhibin A and progesterone concentrations with specific enzyme immunoassays . FSH and LH (1-100 IU/l) increased activin A secretion with 24 h of treatment (to 132% and 253% of control respectively; P<0.05 for both), but their effects were inhibitory in 48-h treatments (26% and 16% decreases respectively; P<0.05 for both) . In the same experiments, FSH and LH increased inhibin A and progesterone secretion after both 24 and 48 h of treatment . 8-BrcAMP (0.1-100 muM) increased activin A in 24- and 48-h experiments (to 206% and 148% of control respectively; P<0.01 for both) . Inhibin A and progesterone secretion were stimulated by 8-BrcAMP time- and dose-dependently . TPA increased activin A secretion dose-dependently (0.1-100 ng/ml) in both 24- and 48-h experiments . At 100 ng/ml concentration, it increased activin A up to 61-fold and inhibin A up to 16-fold of control in 24-h experiments . We conclude that gonadotropins regulate immunoreactive activin A secretion biphasically in cultured human granulosa-luteal cells: initial stimulation is followed by inhibition . In contrast, gonadotropins increase inhibin A and progesterone secretion continuously . Consequently, continuing gonadotropin stimulation leads to a decreasing activin:inhibin ratio, which may have a significant role in the local fine-tuning of ovarian steroidogenesis.

Fertil Steril, 2002 Mar, 77(3), 542 - 7
Estradiol down-regulates MCP-1 expression in human coronary artery endothelial cells; Seli E et al.; OBJECTIVE: To determine whether estrogen down-regulates MCP-1 in vascular endothelial cells . DESIGN: A prospective comparative study . SETTING: Academic research environment . PATIENT(S): Human umbilical vein endothelial cells (n = 3) and human coronary artery endothelial cells (n = 3) obtained from females . INTERVENTION(S): Human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) were grown to preconfluence . Then, they were treated with various concentrations of estradiol (10(-11) M to 10(-7) M) as well as raloxifene (10(-7) M) and tamoxifen (10(-7) M) . MCP-1 in culture media was quantified using an enzyme-linked immunosorbent assay (ELISA) . Cellular ribonucleic acid (RNA) was extracted and Northern blots were hybridized with an oligonucleotide probe complementary to a specific sequence of MCP-1 mRNA . MAIN OUTCOME MEASURE(S): MCP-1 protein and mRNA . RESULT(S): Estrogen treatment did not change MCP-1 expression in HUVEC . On the other hand, in HCAEC, estradiol induced a 30% decrease in mRNA expression and resulted in dose-dependent inhibition of MCP-1 production as detected by ELISA . Raloxifene and tamoxifen also resulted in inhibition of MCP-1 mRNA and protein expression . CONCLUSION(S): Our findings suggest that one of the mechanisms by which estrogen down-regulates atherosclerosis is by suppressing vascular MCP-1 expression, resulting in decreased macrophage recruitment.

Zhonghua Bing Li Xue Za Zhi, 1999 Dec, 28(6), 445 - 9
{Expression of endothelin 1 and tumor necrosis factor alpha in injuried renal tubules and their influences on renal interstitial fibroblasts}; Li L et al.; OBJECTIVE: To study the expression of endothelin 1 (ET-1) and tumor necrosis factor (TNFalpha) in the epithelial cells of normal and injured renal tubules and their influences on renal interstitial fibroblasts . METHODS: Cultivation of renal tubular epithelial cells and renal interstitial fibroblasts and establishment of a renal tubulo-interstitial fibrosis (TIF) animal model; reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemical staining, radioimmunoassay (RIA), double immunohistochemical; and (3)H-TDR incorporation techniques were applied to study the relationship between ET-1, plus TNFalpha and the proliferation of renal interstitial fibroblasts . RESULTS: Expression of ET-1 mRNA and TNFalpha mRNA from renal tubular epithelial cells was obtained (546 bp and 415 bp) respectively as well as the relevant ET-1 and TNFalpha proteins . The concentration of ET-1 and TNFalpha in the culture medium were 1.42 pg/ml and 0.58 ng/ml respectively . The amount of ET-1 and TNFalpha was increased during cell injury and regeneration . In addition, the ratio of (3)H-TDR incorporation and the expression of ET-R mRNA and TNF-R1 mRNA (545 bp and 347 bp) were markedly increased than that of the control group (P < 0.05) when ET-1 or TNFalpha was added in the culture media . CONCLUSION: Injured and regenerated renal tubular epithelial cells are able to synthesize and liberate more ET-1 and TNFalpha than that of the normal renal tubules . ET-1 and TNFalpha are also effective in promoting the proliferation of renal interstitial fibroblasts through ET-R and TNF-R.

Virus Res, 2002 Feb 26, 83(1-2), 71 - 87
Nucleotide sequence and construction of an infectious cDNA clone of an EMCV strain isolated from aborted swine fetus; Kassimi LB et al.; A full-length cDNA clone of an Encephalomyocarditis virus (EMCV) strain (2887A) isolated from aborted swine fetus was constructed and sequenced . Sequence comparison showed more than 99% nucleotide and amino acid sequence identity with two other EMCV strains, EMCV-PV21 and -R . However, the 2887A genomic sequence showed only about 84% nucleotide identity and 96% amino acid identity with EMCV-B, -D and -PV2 variants . RNA synthesized by in vitro transcription of this cDNA clone was infectious upon transfection of BHK21 cells, as shown by cytopathic effects and identification by neutralization test, and by propagation of the virus released into the culture media . The transcript RNA led to the production of infectious particles despite the presence of two nongenomic nucleotide residues at the 5' end, the short poly(C) tract (C(10)TCTC(3)TC(10)), the short poly(A) tail (7A), and the presence of six nongenomic nucleotides at the 3' end . The rescued virus was also found to be highly pathogenic for mice by intra-peritoneal inoculation producing a fatal disease indistinguishable from that of wild-type virus . An important finding concerning the molecular basis of infectivity was that the in vitro synthesized EMCV RNA transcript is infectious, although it contains a very short poly(A) . The availability of the infectious cDNA clone of the reproductive failure strain of EMCV should prove to be useful for studying the molecular basis of the pathogenicity of EMCV in pig.

Biochem J, 2002 Mar 1, 362(Pt 2), 465 - 72
The mechanism of aggrecan release from cartilage differs with tissue origin and the agent used to stimulate catabolism; Sztrolovics R et al.; The mechanisms of aggrecan degradation in adult human articular, adult bovine nasal and fetal bovine epiphyseal cartilage in response to either interleukin-1beta (IL-1beta) or retinoic acid were compared using an explant culture system . Bovine nasal cartilage cultured with either IL-1beta or retinoic acid exhibited significant release of glycosaminoglycan (GAG) . For both factors, aggrecan proteolysis occurred predominantly at the 'aggrecanase' site, with no evidence for the action of matrix metalloproteinases, and resulted in the appearance of the corresponding G1 fragment in tissue extracts and in culture media . In human cartilage, little effect of IL-1beta was seen, but abundant release of GAG occurred in the presence of retinoic acid, with evidence of aggrecanase action . Treatment of fetal epiphyseal cartilage with retinoic acid resulted in significant GAG release, whereas treatment with IL-1beta did not . In the retinoic acid-treated tissue, however, no evidence for the cleavage of aggrecan in the interglobular region was apparent . Thus, in the fetal system, agents in addition to aggrecanase and matrix metalloproteinases appear to be active . Taken together, these data demonstrate that the pathways utilized for aggrecan catabolism may vary between different cartilages for a given stimulatory agent, and that, for a given tissue, different factors may elicit aggrecan release via different pathways.

Arch Dermatol . 1965 Jul;92(1):103.
"Brush" technique in animals . Finding contact sources of fungus diseases; Goldberg HC; A technique is described for collecting specimens of hairs and scales from animals by means of a brush to help in the diagnosis of fungus diseases . The brush is then used to implant such materials on special culture media for the growth and identification of fungus organisms . This method offers a better chance to find fungus organisms than the previous method of plucking, which was like looking for a needle in a haystack . The special media makes possible the rapid diagnosis of the presence of pathogenic fungi.

Brain Res Dev Brain Res, 2002 Jan 31, 133(1), 27 - 35
Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide; Gayle DA et al.; Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc) . Although the exact mechanisms responsible for this cell loss are unclear, emerging evidence suggests the involvement of inflammatory events . In the present study, we characterized the effects of the proinflammatory bacteriotoxin lipopolysaccharide (LPS) on the number of tyrosine hydroxylase immunoreactive (THir) cells (used as an index for DA neurons) in primary mesencephalic cultures . LPS (10-80 microg/ml) selectively decreased THir cells and increased culture media levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as well as nitrite (an index of nitric oxide (NO) production) . Cultures exposed to both LPS and neutralizing antibodies to IL-1beta or TNF-alpha showed an attenuation of the LPS-induced THir cell loss by at least 50% in both cases . Inhibition of the inducible form of nitric oxide synthase (iNOS) by L-NIL did not affect LPS toxicity, but increased the LPS-induced levels of both TNF-alpha and IL-1beta . These findings suggest that neuroinflammatory stimuli which lead to elevations in cytokines may induce DA neuron cell loss in a NO-independent manner and contribute to PD pathogenesis.

J Immunoassay Immunochem, 2002, 23(1), 33 - 48
Immunoassay for measuring the heparin-binding growth factors HARP and MK in biological fluids; Soulie P et al.; Heparin-affin regulatory peptide (HARP) and Midkine (MK) belong to a family of growth/differentiation factors that have a high affinity for heparin . The involvement of these molecules in various proliferative diseases prompted us to develop an assay for measuring the concentrations of these factors in biological fluids and culture media . This report describes an immunoassay that uses only commercially available materials, based on the high affinity of certain molecules for heparin . It consists of adsorbing heparin-BSA covalent complexes to microtiter plate wells and to quantify the heparin bound HARP or MK by using appropriate antibody . The method is specific and measures concentrations ranging from 40-1200 pg/mL HARP and from 25-1200 pg/mL MK and various parameters are investigated . The within-assay coefficient of variation was less than 5% for both assays . The method was checked by measuring the concentrations of these growth factors in the sera of healthy humans and in patients with cancer . As previously reported, we confirmed that the serum concentrations of MK are higher in patients with tumours (n = 139) than in controls (n = 19) . The synthesis of HARP and MK by various cells in culture was also analysed.

Hum Fertil (Camb), 2000, 3(4), 229 - 237
Blastocyst culture: toward single embryo transfers; Gardner DK; The routine culture and transfer of viable human blastocysts has been made possible by the development of sequential culture media, formulated to account for the changes in nutrient requirements of the embryo as it develops and differentiates . Resultant implantation rates of blastocysts transferred on day 5 are significantly higher than those obtained by the transfer of cleavage stage embryos transferred on day 2 or day 3 within the same programme . As a direct result of this increase in implantation rate, fewer blastocysts than cleavage stage embryos need to be transferred to obtain acceptable pregnancy rates, thereby reducing the incidence of multiple gestations . Blastocysts developed in sequential culture media are readily cryopreserved . The efficiency of in vitro fertilization (IVF) in a general patient population can be calculated using a model that takes into account the number of embryos transferred and cryopreserved, together with their respective implantation rates . Blastocyst transfer is associated with about a 20% increase in the efficiency of IVF compared with the transfer of cleavage stage embryos on day 3 . The development of a suitable scoring system has enabled identification of those blastocysts with the highest developmental potential (70% implantation rate) . The culmination of this work should be the move to the transfer of a single blastocyst for a significant number of patients.

Respirology, 2001 Dec, 6(4), 271 - 9
Infection of replication-deficient adenoviral vector enhances interleukin-8 production in small airway epithelial cells more than in large airway epithelial cells; Kodama Y et al.; OBJECTIVE: In clinical trials or experiments of gene therapy, airway administration of an adenoviral-based vector (E1A-deleted) elicits a dose-dependent inflammatory response with limitation in the duration of transgene expression . The purpose of this study was to evaluate the possibility that the adenoviral-based vector directly enhances IL-8 production independent of adenoviral E1A in normal human airway epithelial cells and to examine the different responses between primary human bronchial epithelial cells (HBE) and primary human small airway epithelial cells (HSAE) in production of IL-8 following exposure to an adenovirus vector . METHODOLOGY: Interleukin (IL)-8 levels were evaluated in the culture medium from HBE and HSAE treated with increasing doses of E1A-deleted adenoviral vector contained the Escherichia coli LacZ reporter gene (AdCMVLacZ) . To clarify the mechanism of enhancing IL-8 production in airway epithelial cells by infection with adenovirus vector, alphavbeta5 agonistic antibody as an analogue of adenoviral capsid and adenoviral capsid vector denatured by exposure to ultraviolet (UV) light were used in the present study . RESULTS: Inoculation of HBE with AdCMVLacZ at a multiplicity of infection (MOI) of between 1 and 200 resulted in a dose-dependent expression of LacZ, and maximal expression was observed at a MOI of 100 . In contrast, inoculation of HSAE with AdCMVLacZ resulted in maximum expression of LacZ at a MOI of 10 . Interleukin-8 levels in culture media from the same experiments revealed significantly greater production of IL-8 in HSAE inoculated with AdCMVLacZ at a MOI of 50, compared to HBE under the same conditions . The capsid-denatured adenoviral vector did not enhance IL-8 production, and alphavbeta5 agonistic antibody induced IL-8 enhancement . CONCLUSION: These results suggest that the adenoviral vector directly induces the expression of airway epithelial inflammatory cytokines in the pathogenesis of inflammation and that small airway cells have a greater affinity for adenovirus than other airway epithelial cells.

J Surg Res, 2002 Feb, 102(2), 57 - 62
Dexamethasone inhibits vascular smooth muscle cell migration via modulation of matrix metalloproteinase activity; Pross C et al.; BACKGROUND: Dexamethasone (DEX) has been shown to inhibit development of neointimal hyperplasia in rats . We hypothesize that DEX inhibits neointimal hyperplasia by altering matrix metalloproteinase (MMP) activity, resulting in inhibition of smooth muscle cell migration . METHODS: Rat aortic smooth muscle cells (RASMC) were harvested and cultured for two to four passages . A migration assay was performed in a Boyden chamber with chemoattractant (platelet-derived growth factor) and varying concentrations of DEX (10(-9) to 10(-5) M) . The number of migrated cells was counted under light microscopy . Zymography was performed on culture media to assess MMP activity, and Western blotting was performed to assay MMP and levels of tissue inhibitors of MMPs (TIMPs) . RESULTS: DEX progressively inhibited RASMC migration in a dose-dependent fashion . This effect was statistically significant for concentrations of 10(-7) to 10(-5) M (P < 0.0005) . Zymography showed that DEX inhibits MMP-2 activity in a dose-dependent manner . Western blots indicated that total MMP-2 secretion was inhibited and that TIMP-2 secretion was increased by DEX . CONCLUSIONS: DEX inhibits platelet-derived growth factor-induced migration of RASMCs and MMP-2 activity in vitro . Our data suggest that DEX suppresses MMP activity and secretion, resulting in the inhibition of smooth muscle cell migration . This may explain the mechanism by which DEX inhibits neointimal hyperplasia . (c)2001 Elsevier Science.

Osteoarthritis Cartilage, 2001 Nov, 9(8), 712 - 9
Human chondrocyte apoptosis in response to mechanical injury; D'Lima DD et al.; OBJECTIVE: The effect of mechanical injury on chondrocyte viability and matrix degradation was studied . It was proposed that mechanical injury to human cartilage explants results in chondrocyte apoptosis with associated loss of glycosaminoglycans . DESIGN: Full thickness human cartilage explants, 5 mm in diameter were subjected to a single static mechanical stress of 14 MPa for 500 ms under radially unconfined compression . Glycosaminoglycan (GAG) release and percentage of cells undergoing apoptosis were measured at 96 h after injury . To establish the time course of apoptosis, explants were subjected to 30% strain and cultured for varying intervals up to 7 days after injury . A group of loaded explants were also treated with the broad spectrum caspase inhibitor z-Vad.fmk after injury . RESULTS: Internucleosomal DNA fragmentation as one indicator of apoptosis was observed in 34% (S.D.+/-11) of chondrocytes at 96 h in response to mechanical loading at 14 MPa, compared to 4% (S.D.+/-2) in the non-loaded explants . Evidence for cell death induction via apoptosis was also obtained by electron microscopy and caspase cleavage of cytokeratin . GAG release was also higher for the loaded explants, mean 1.9% (S.D.+/-0.14) of total GAG content, compared to control explants, mean 0.8% (S.D.+/-0.28) . The percentage of apoptotic cells also correlated with the level of GAG release into the culture media . The percentage of apoptotic chondrocytes demonstrated a progressive increase from 6 h to 7 days post-injury . When loaded explants were cultured in z-Vad.fmk after injury, a 50% reduction in apoptosis rates was seen . CONCLUSIONS: These results demonstrate that mechanical injury induces chondrocyte apoptosis and release of GAG from the matrix . The time course suggests that a therapeutic window may exist where apoptosis could be inhibited . This potentially identifies a new approach to chondroprotection .

Gut, 2002 Mar, 50(3), 392 - 401
Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor; Nagy N et al.; BACKGROUND AND AIMS: Galectins are beta-galactoside binding proteins . This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells . In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features . This evident gap has prompted us to extend galectin analysis beyond the two prototypes . The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts . The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines . METHODS: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with alpha- and beta-D-galactose, alpha-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy . The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction . In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy . In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy . RESULTS: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue . Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0) . The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo . Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate . In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo . CONCLUSIONS: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts . In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin . Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Hybrid Hybridomics, 2001, 20(5-6), 361 - 8
The nucleotide sequence of dinitrophenyl-specific IgE and Fc(epsilon)RI alpha-subunit obtained from FE-3 hybridoma cells; Tokeshi Y et al.; FE-3 cells were established by Hanashiro et al . by hybridizing mouse myeloma cells (Sp2/0-Ag14/SF) with rat spleen cells that were freshly isolated from Brown-Norway rats sensitized with DNP-As . FE-3 cells can constitutively secrete IgE without stimulation by cytokines . Our preliminary experiments demonstrated that the IgE secretion was decreased at 3 days after start of culture and the addition of exogenous IgE into culture media depressed the secretion of IgE . Thus, we hypothesized that the IgE production in FE-3 cells may be regulated by a signal transduction through the binding of IgE to its high affinity receptor (Fc(epsilon)RI) or to an IgE binding protein on the cell surface . In this study, we aimed to identify the nucleotide sequence of IgE FE-3 and compared with those of mouse IgE and IgE IR162 to find a structural heterogeneity in the Fc region of IgE FE-3 . We also tested if the mRNA of Fc(epsilon)RI was expressed in FE-3 cells using the reverse transcriptase-polymerase chain reaction (RT-PCR) method with the combination of sequencing analysis . Consequently, the cDNA sequence of IgE FE-3 was identical to that of the CH3 and CH4 domains in the epsilon-chain of rat IgE IR162, whereas the cDNA of Fc(epsilon)RI was identical to that of mouse, suggesting that the genes of IgE FE-3 and Fc(epsilon)RI was derived from that of rat spleen cells and mouse myeloma cells, respectively.

New Microbiol, 2002 Jan, 25(1), 57 - 64
Bioconversion of poultry wastes I-factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi; Abdel-Faftah GM et al.; The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus . A . flavus and Trichoderma sp . was at 30 degrees C . The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp . The optimum pH was at 6.4 for A . terreus and pH 6.6 for both A . flavus and Trichoderma sp . The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste . The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A . terreus, A . flavus and Trichoderma sp., respectively . Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi . The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, but did not significantly increase uricase production.

Environ Toxicol Chem, 2002 Feb, 21(2), 404 - 12
Influence of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid pH buffer on the biological response of marine algae; Vasconcelos MT et al.; The N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) is extensively used as pH buffer in culture media for testing chemicals . However, this study demonstrates that 0.01 M HEPES significantly reduces the rate of Cu, Pb, and Cd binding to Porphyra spp . and Enteromorpha spp . marine macroalgae . The HEPES also decreased the accumulation of Cu, Pb, and Cd but not Hg by these macroalgae . Both the extracellular adsorption and the intracellular uptake of the metals were influenced by HEPES to a similar extent . The HEPES also promoted the release of exudates by the algae, and these exudates form very stable complexes with Cu (and probably with other trace metal ions) . The HEPES interference varied with the nature of the metal, the macroalga, and the season . The presence of 0.01 M HEPES in seawater cultures of the Emiliania huxleyi (a microalga) also interfered with E . huxleyi growth, liberation of Cu-complexing organic ligands, and Cu uptake . The HEPES, which displays surface activity, may facilitate the binding of metals to the algae for an initial exposure period . The metal taken up appears to stimulate the liberation of exudates that subsequently control the bioavailability of the metals and therefore metal uptake . Because HEPES can control the uptake of trace metals by algae and the production of organic ligands, the results obtained in cultures containing the HEPES pH buffer can be influenced by this component of the media.

J Clin Endocrinol Metab, 2002 Feb, 87(2), 700 - 8
Cortisol/progesterone antagonism in regulation of 15-hydroxysteroid dehydrogenase activity and mRNA levels in human chorion and placental trophoblast cells at term; Patel FA et al.; PGs mediate parturition events . 15-Hydroxyprostaglandin dehydrogenase (PGDH) catalyzes the first step in the metabolism of PGs to render them inactive . We have reported previously that cortisol (F) decreases PGDH activity and progesterone (P(4)) maintains PGDH in human chorion and placenta at term . To study the interaction of P(4) and F on the regulation of PGDH, we treated chorion and placental trophoblast cells in culture with combinations of F, dexamethasone, P(4), trilostane, and medroxyprogesterone acetate (MPA) . Following a 24-h steroid treatment period and 4-h PGF(2alpha) challenge, culture media and cells were collected for measurement of PGF(2alpha) levels and PGDH mRNA by RIA and Northern blotting analysis . F and dexamethasone decreased PGDH activity and mRNA levels . Exogenous P(4) did not significantly alter PGDH activity or mRNA levels; however, MPA significantly stimulated PGDH activity . Trilostane decreased P(4) production by more than 90% and also decreased PGDH activity and expression . Coincubation with P(4) or MPA reversed trilostane inhibition of PGDH, consistent with a stimulatory role for endogenous P(4) on PGDH . MPA significantly reversed F inhibition of PGDH activity and mRNA levels . In the presence of trilostane, P(4) at equimolar concentration to F reversed F inhibition of PGDH mRNA levels . These findings suggest that F may be acting as an endogenous inhibitor of P(4) action in the regulation of PGDH at term.

J Endocrinol, 2002 Feb, 172(2), 333 - 44
Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells; S-L Zhang et al.; Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development . We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway . We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes . Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration . Kidney proximal tubules were isolated and cultured in either normal glucose (i.e . 5 mM) or high glucose (i.e . 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression . Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively . Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit . Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs . In contrast, it failed to affect these parameters in diabetic rat RPTCs . In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.

J Nutr Biochem, 2002 Jan, 13(1), 55 - 63
Polyunsaturated fatty acids downregulate the low density lipoprotein receptor of human HepG2 cells; Pal S et al.; The aim of the study was to investigate the effect of different fatty acids on the low density lipoprotein (LDL) receptor of cultured human liver HepG2 cells . Previous studies investigating the effect of fatty acids on LDL expression have reported conflicting findings and are limited to measurements of LDL receptor binding activity . Therefore, this study is unique in that the relative effects of different fatty acids on the LDL receptor were investigated at three different stages of expression: 1) functional cellular LDL binding activity, 2) amount of LDL receptor protein and 3) LDL receptor mRNA level . The HepG2 cells were incubated for 24 hr with either 100 &mgr;M palmitic, oleic, linoleic or eicosapentaenoic acid (EPA) . The measurement of LDL receptor binding activity was with colloidal gold-LDL conjugates, cellular LDL receptor protein was by western blotting and LDL receptor mRNA by Southern blotting of reverse-transcribed, polymerase chain reaction-amplified cDNA . The LDL receptor binding activity, protein and mRNA levels decreased as the degree of unsaturation of the fatty acids increased (palmitic acid greater-than-or-equal oleic acid > linoleic acid > EPA) and the inverse relationship held whether or not cholesterol was included in the culture media . The relative differences were very similar for the three stages of expression indicating that modulation of the LDL receptor by the fatty acids occurred at the level of gene transcription . The increased susceptibility to oxidation of the polyunsaturated fatty acids was unlikely to be a factor in the effect because EPA and linoleic acid (250 &mgr;M) still downregulated the LDL receptor in the presence of the antioxidant vitamin E (50 &mgr;M) . In conclusion, the polyunsaturates, linoleic acid and EPA, effectively downregulated the LDL receptor of HepG2 cells compared to palmitic acid . The effects of these fatty acids were observed at the level of LDL receptor binding activity, protein and mRNA, strongly suggesting that the fatty acid effects were at the level of gene transcription.

Reprod Fertil Dev, 2001, 13(5-6), 361 - 5
Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro; Gutierrez-Adan A et al.; The effect of glucose in the medium used during in vitro culture on the sex ratio of bovine blastocysts derived from in-vitro-matured and in-vitro-fertilized oocytes was evaluated . Oocytes were matured and inseminated with mixed sperm from three bulls and were cultured in vitro in modified synthetic oviducal fluid medium with 10% fetal calf serum, with or without glucose supplementation . The overall rate of cleaved embryos that developed to expanded blastocyst in the medium without glucose (27.0%) was significantly greater (P < 0.05) than the percentage observed when embryos were cultured in medium with glucose (17.5%) . Analysis of variance was performed to analyse the effect of glucose on the proportion of male embryos reaching the blastocyst stage (or arrested at the morula stage) during Days 7 to 10 . Regardless of the presence or absence of glucose in the medium, significantly (P < 0.05) more male than female embryos were harvested as expanded blastocysts on Day 7 and on Day 8 of culture . On Days 9 plus 10 of culture, a sex ratio imbalance only occurred in the absence of glucose in the culture medium (P < 0.05) . Glucose did not produce any significant effect on the sex ratio of the overall number of expanded blastocysts harvested by Day 10 of in vitro culture . However a significantly greater proportion of females (P < 0.01) were found among those embryos that developed only to the morulae stage after 10 days in vitro . These results show that glucose supplementation of culture media produces a preferential loss of female embryos during culture to the blastocyst stage.

Reprod Fertil Dev, 2001, 13(5-6), 355 - 60
Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro; Gupta PS et al.; In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning . In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V, Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated . Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL(-1) in presence or absence of steer serum (10%) for 24 h in a CO2 incubator . The maturation rate was assessed on the basis of degree of expansion of cumulus cells . The matured oocytes were inseminated with 9-10 x 10(6) spermatozoa mL(-1) in Brackett and Oliphant medium and the cleavage rate was recorded 40-42 h after insemination . Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates . The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days . Maturation, fertilization, cleavage and embryonic development were significantly higher (P < 0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL(-1) . It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.

Osteoarthritis Cartilage, 2002 Jan, 10(1), 5 - 12
Inducible nitric oxide expression in equine articular chondrocytes: effects of antiinflammatory compounds; Tung JT et al.; OBJECTIVE: To determine the effects of recombinant equine IL-1beta and a number of antiinflammatory compounds on the expression and activity of inducible nitric oxide synthase (iNOS) in cultured equine chondrocytes . DESIGN: RT-PCR methods were used to amplify a portion of the equine iNOS message to prepare an RNA probe . Northern blot analysis was used to quantify the expression of iNOS in first passage cultures of equine articular chondrocytes propagated in the presence or absence of recombinant equine interleukin-1beta (reIL-1beta), dexamethasone (DEX), polysulfated glycosaminoglycan (PSGAG), hyaluronan (HA), and phenylbutazone (PBZ), each at concentrations of 10 and 100 microg/ml . Nitrite concentrations in conditioned media of similarly treated cells were used to quantify iNOS activity . RESULTS: Recombinant equine IL-1beta increased the expression of iNOS in a dose-dependent manner . This result was paralleled by an increased concentration of nitrite in the culture media of reIL-1beta-treated cells . DEX and PSGAG significantly reduced iNOS gene expression and media supernatant nitrite concentrations in cytokine-stimulated cultures . HA and PBZ had no consistent effect on the expression of iNOS and did not significantly influence nitrite content of conditioned media . CONCLUSIONS: NO is considered an important mediator in the pathophysiologic processes of arthritis and an inducible NOS is expressed by equine chondrocytes . Pre-translational regulation of the iNOS gene by DEX and PSGAG appears to contribute to the cartilage-sparing properties of these compounds .

J Eukaryot Microbiol, 2001 Nov-Dec, 48(6), 616 - 21
Correlation between in vitro and in vivo infectivity of Leishmania infantum clones; Mendez S et al.; Eleven clones of a single strain of Leishmania infantum (MCAN/ES/88/ISS441, Doba) were analyzed for biological behavior in vivo and in vitro . Different clones showed differences in growth dependent upon the two culture media employed . All clones displayed only slight differences in H2O2 and NaNO2 sensitivity compared to the original strain, whereas in vitro infectivity for mouse peritoneal macrophages differed significantly among the clones . In vivo infections in hamsters correlated strongly with in vitro infectivity . The phenotypic differences found suggest a polyclonal structure for the Leishmania infantum strain studied.

Nitric Oxide, 2002 Feb, 6(1), 73 - 8
Environmental pH regulates LPS-induced nitric oxide formation in murine macrophages; Huang CJ et al.; This study was designed to determine how pH affects nitric oxide (NO) formation induced by lipopolysaccharide (LPS) in cultured murine macrophages (RAW 264.7) . The initial pH of LPS-containing culture media was adjusted to one of eight values (6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and 8.2) . After exposure to LPS for eighteen hours, the cultures were harvested for analysis of mRNA, protein, and nitrate/nitrite (stable by-products of NO) . Analyses for these substances were performed using semiquantitative RT-PCR, immunoblotting, and colorimetric Griess assays, respectively . We found that acidic culture media favored expression of inducible nitric oxide synthase (iNOS) mRNA . However, alkaline media favored expression of iNOS protein . Our findings suggest that post-transcriptional mechanisms predominate over transcriptional ones in order to regulate pH-mediated effects on NO formation by murine macrophages . The optimal pH for NO formation by iNOS was found in our study to be around 7.2.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Jan 25, 766(2), 295 - 305
Determination of lonazolac and its hydroxy and O-sulfated metabolites by on-line sample preparation liquid chromatography with fluorescence detection; Friedrich G et al.; A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described . The procedure employs on-line sample enrichment using a BioTrap 500 MS (20 x 4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C18-HT (100 x 3 mm I.D., 3.5 microm particles) analytical column in the back-flush mode . Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission . Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC-MS-MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode . Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2 +/- 3.5, 96.7 +/- 2.2, and 100.9 +/- 3.5% . The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 microl . Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10-600 ng/ml . The mean values of the coefficients of variation (CV) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46 +/- 1.15, 3.94 +/- 2.13 and 4.79 +/- 2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate . The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.

Biotechnol Prog, 2002 Jan-Feb, 18(1), 155 - 8
Specific effects of synthetic oligopeptides on cultured animal cells; Franek F et al.; Synthetic oligopeptides, tri- to pentaglycine and tri- and tetraalanine, were found to enhance viable cell density and culture viability when applied at concentrations higher than milllimolar to the cultures of a model hybridoma line . Oligoalanines, in addition, enhanced monoclonal antibody yields . Oligoglycines promoted solely the cell growth, unless the batch culture was fed with a medium concentrate . Examination of the effects of various tripeptides composed of glycine, alanine, serine, threonine, lysine, and histidine showed that some of the peptides promoted the growth of the culture, while other peptides suppressed the growth and enhanced the monoclonal antibody yield . Determination of the levels of amino acids and peptides in culture media indicated that the observed changes of culture parameters were caused by intact peptide molecules, rather than by amino acids liberated from the peptides by enzymic cleavage.

Fertil Steril, 2002 Feb, 77(2), 392 - 5
Secretion of keratinocyte growth factor by cultured human endometrial stromal cells is induced through a cyclic adenosine monophosphate-dependent pathway; Nasu K et al.; OBJECTIVE: To evaluate the effects of known modulators of endometrial function on the production of keratinocyte growth factor by endometrial stromal cells . DESIGN: The effects of dibutyryl-cyclic adenosine monophosphate (db-cAMP), 12-O-tetradecanoylphorbol 13-acetate (TPA), ethinyl estradiol-17alpha (EE), and medroxyprogesterone acetate (MPA) on the secretion of keratinocyte growth factor by endometrial stromal cells were investigated . SETTING: Research laboratory at a university medical school . PATIENT(S): Eleven endometrial specimens in the late proliferative phase . INTERVENTION(S): Endometrial stromal cells were incubated for 24 hours with db-cAMP, TPA, EE, or MPA . MAIN OUTCOME MEASURE(S): The concentration of keratinocyte growth factor in the culture media was measured using an ELISA . RESULT(S): Small amounts of keratinocyte growth factor were detected in the culture media of unstimulated endometrial stromal cells . The production of keratinocyte growth factor by endometrial stromal cells was stimulated with db-cAMP in a dose-dependent manner . The stimulatory effect of db-cAMP was inhibited by Rp-adenosine 3',5'-cyclic monophosphothioate . None of TPA, EE, nor MPA affected the keratinocyte growth factor production by these cells . CONCLUSION(S): These results suggest that a cAMP-dependent pathway may play an important role in the regulation of keratinocyte growth factor production by endometrial stromal cells . Keratinocyte growth factor secreted by endometrial stromal cells may be involved in the regeneration of the endometrium during the normal menstrual cycle and early pregnancy.

World J Gastroenterol, 1998 Aug, 4(4), 329 - 331
Effect of endotoxin on fibronectin synthesis of rat primary cultured hepatocytes; Jia JB et al.; AIM:To investigate the effect of endotoxin on liver fibrosis and further define the role of hepatocytes in production of fibronectin in primary liver cell culture by endotoxin.METHODS:After isolation and seeding of hepatocytes, the obtained cells were added to various doses (0, 5, 10, 15 and 20mg/L) of LPS treated culture media . The cells were collected and counted at various periods (0, 12, 24, 48, 72, 96, 120h).The concentrations of fibronectin were tested by electrophoresis . RESULTS:The fibronectin levels tended to increase with prolongation of culture time . There was a sharp increase after 72h in 10 or 15 LPS treated group . The peak level of fibronectin was above 20mg/L . However, cell proliferation was inhibited during the course.Cell number of untreated control group (4.6 +/- 0.1 10(6)) was about three fold that of 20 LPS treated group (1.6 +/- 0.2 10(6)) at 120h.CONCLUSION:Hepatocytes have a potent ability to produce fibronectin stimulated by endotoxin, suggesting that hepatocytes might participate in the process of liver fibrosis.

World J Gastroenterol, 1998 Apr, 4(2), 121 - 124
Serum deprivation enhances DNA synthesis of human hepatoma SMMC7721 cells; Jiang SM et al.; AIM:To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC-7721 cells.METHODS:Human hepatoma SMMC-7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS)in 5% CO(2) incubator at 37&mgr; for 24h, and culture media were replaced to serum-free or different serum FCS levels (2.5%, 5%, 10%, 20% and 25%) . Six h,12h,18h and 24h after the culture,the cells were incorporated &mgr;(3)H&mgr;-TdR for 4h . At last &mgr;(3)H&mgr;-TdR incorpor-ation was detected with liquid scintillation counting.RESULTS:DNA synthesis of SMMC-7721 cells could be sharply stimulated by short-time (6h) serum deprivation (the cpm value of (3)H-TdR incorporation of cells in serum-free was 39.32-fold higher than cells in 25% serum),and the incorporation of (3)H-TdR was negatively related to the serum levels.Longer-time serum starvation (12h, 18h and 24h) also greatly stimulated DNA synthesis, although the cpm value of (3)H-TdR incroporation was less than that in 6h serum deprivation . Morphology of cells cultured in different serum levels also showed significant difference.CONCLUSION:Compared with other cell lines such as BEL7404 and Swiss 3T3,human hepatoma SMMC-7721 cells had different response to the serum deprivation.Short-time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC-7721 cells.Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC-7721 cells as a model.

Mol Hum Reprod, 2002 Feb, 8(2), 176 - 80
Expression of interferon-gamma-inducible protein-10 in human endometrial stromal cells; Kai K et al.; Human endometrial stromal cells (ESC) can produce a variety of chemokines, especially after inflammatory stimulation . Interferon-gamma-inducible protein-10 (IP-10) is a potent chemoattractant for lymphocytes, and belongs to the family of non-ELR CXC chemokines . The expression of IP-10 in ESC after stimulation with interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), or lipopolysaccharide (LPS) was evaluated using an enzyme-linked immunosorbent assay and Northern blot analysis . A small amount of IP-10 protein was detected in the culture media of unstimulated ESC . The expression of IP-10 mRNA was detected in ESC . IFN-gamma, IL-1 beta, TNF-alpha and LPS significantly stimulated the expression of IP-10 mRNA and protein in ESC . These results suggest that the production of IP-10 by ESC is regulated by inflammatory mediators . The modulation of IP-10 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating leukocyte trafficking in the endometrium.

Perfusion, 2002 Jan, 17(1), 15 - 21
Soluble VCAM-1 is a very early marker of endothelial cell activation in cardiopulmonary bypass; Andresen TK et al.; Cardiopulmonary bypass causes a systemic inflammatory reaction, which leads to endothelial activation with increased expression of adhesion molecules . The study aim was to test whether activated endothelial cells secrete measurable amounts of soluble adhesion molecules during the time course of routine heart surgery, and whether these markers correlate with cellular activation responses . Endothelial cells from human umbilical cords were cultured by standard methods and stimulated with endotoxin . After 2 h, the expression of membrane-bound E-selectin on the cells had increased significantly (p=0.04), whereas soluble VCAM-1 (sVCAM-1) had increased significantly in the culture media (p=0.03) . In agreement with these findings, sVCAM-1 increased from 399 ng/ml (median) to 581 ng/ml within 3 h postoperatively in sera from 11 patients undergoing open heart surgery (p=0.003) . sVCAM-1, therefore, may be suitable as an early marker of endothelial activation related to the systemic inflammation after open heart surgery . The clinical significance of sVCAM-1 measurements must be further evaluated in future patient studies.

Ginecol Obstet Mex, 2001 Oct, 69, 375 - 8
{Assessment of the penetration of culture media in uterine cavity as an effect of cervical lavage before embryo transfer: prospective and observational study of samples obtained in hysterectomy}; Kably A et al.; Cervical lavage used to remove and cleaning some of the scale elements as well as cervical mucous during embryo transfer has been a regular practice in many reproductive centers worldwide . However, the microenvironment influence using these techniques will or not be detrimental in the embryo development . Under this issue, a prospective study was doing in 16 patients (underwent hysterectomy) . A cervical lavage was performed previous to the procedure similar to the embryo's transfer step, subsequently cervical invasion was checking . The age was 36.4 +/- 8.6 years, preoperatory diagnosis was abnormal uterine bleeding (n = 4), myomata (n = 4), adenomiosis (n = 4), endometrial polyp (n = 3) and chronic pelvic pain (n = 1) . Uterine weight was 127.5 +/- 55.4 g with a surgical time of 48.8 +/- 12.5 minutes . Medium in the uterine cavity was founded in only one case . We believe that cervical lavage is a secure technique in embryo transfer.

Prog Lipid Res, 2002 May, 41(3), 240 - 53
In vitro studies on the relationship between polyunsaturated fatty acids and cancer: tumour or tissue specific effects?
Diggle CP.
In vitro cell culture experiments have lead to the consensus in the literature that certain PUFAs have a selective cytotoxic or anti-proliferative effect on tumour cells and a minimal, or no effect on normal cells . Re-examination of key publications showed that when normal cells were used for comparison, they were generally not from the same cell, tissue, or species type as the tumour cells . Recently, investigations have included more appropriate normal control cells, and though tumour specific cytotoxic/anti-proliferative PUFA effects are found in some cell types, in others the normal cells are more sensitive . Cell type differences were found in the relative ability of individual PUFAs to act . However, within a cell type differences in susceptibility were influenced by grade and stage of tumour, immortalisation and tumourigenic status, cell culture media and cell plating density . Together these results suggest that the consensus is not valid, and that susceptibility to PUFA is cell type specific, and alters during neoplastic progression . Furthermore, the cytotoxic/anti-proliferative effect induced by both n-3 and n-6 PUFAs on a wide variety of cell types, associated with an increase in lipid peroxidation in vitro, cannot account for the in vivo data on the relationship between dietary fat and certain cancers . However, the effects of PUFAs and their metabolites on cell signalling pathways may explain the in vivo data.

Diabetes, 2002 Feb, 51(2), 356 - 65
Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes; Alexander AM et al.; Indoleamine 2,3-dioxygenase (IDO) catalyzes the breakdown of the amino acid tryptophan into kyneurenine . It has been shown that IDO production by placental trophoblasts prevents the attack of maternal T-cells activated in response to the paternal HLA alleles expressed by the tissues of the fetus . In this article, we show that adenoviral gene transfer of IDO to pancreatic islets can sufficiently deplete culture media of tryptophan and consequently inhibit the proliferation of T-cells in vitro . Experiments in vivo have also demonstrated that transplantation of IDO-expressing islets from prediabetic NOD mouse donors into NODscid recipient mice is associated with a prolongation in islet graft survival after adoptive transfer of NOD diabetogenic T-cells . This protection is attributed to the depletion of tryptophan at the transplantation site beneath the kidney capsule . These results suggest that local modulation of tryptophan catabolism may be a means of facilitating islet transplantation as a therapy for type 1 diabetes.

Anal Biochem, 2002 Feb 1, 301(1), 21 - 6
Concurrent quantification of quinolinic, picolinic, and nicotinic acids using electron-capture negative-ion gas chromatography-mass spectrometry; Smythe GA et al.; Quinolinic, picolinic, and nicotinic acids and nicotinamide are end products of the kynurenine pathway from l-tryptophan and are intermediates in the biosynthesis of nicotinamide adenine dinucleotide . These compounds are involved in complex interrelationships with inflammatory and apoptotic responses associated with neuronal cell damage and death in the central nervous system . To facilitate the study of these compounds, we have utilized gas chromatography-mass spectrometry in electron capture negative ionization mode for their concurrent trace quantification in a single sample . Deuterium-labeled quinolinic, picolinic, and nicotinic acids were used as internal standards and the compounds were converted to their hexafluoroisopropyl esters prior to chromatography . Nicotinamide was readily quantified after conversion to nicotinic acid using gas-phase hydrolysis-a process which did not affect the deuterated internal standards . The on-column limit of quantification was less than 1 fmol for each of the analytes and calibration curves were linear . A packed column liner was used in the gas chromatograph inlet to effectively eliminate sample interference effects in the analysis of trace (femtomolar) levels of quinolinic acid . The method enables rapid and specific concurrent quantification of quinolinic, picolinic, and nicotinic acids in tissue extracts and physiological and culture media.

J Periodontol, 2001 Dec, 72(12), 1685 - 94
Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture; Kut-Lasserre C et al.; BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs) . They can also respond to growth factors and cytokines . In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable {ASF} or soy unsaponifiable {SSF}) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts . METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0 . 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta) . MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis . TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis . RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF . The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml) . A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations . Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF . As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen . ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml . CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.

Microsc Res Tech, 2002 Jan 15, 56(2), 101 - 12
Ciliary neurotrophic factor increases the survival of magnocellular vasopressin and oxytocin neurons in rat supraoptic nucleus in organotypic cultures; Rusnak M et al.; Organotypic cultures of the rat hypothalamus are very useful models for the long-term study of parvocellular vasopressin (VP) neurons in the paraventricular (PVN) and suprachiasmatic (SCN) nuclei . However, they do not preserve significant numbers of VP magnocellular neurons (VP-MCNs) in either the PVN or the supraoptic nucleus (SON) . Vutskits et al . {(1998) Neuroscience 87:571-582} reported that ciliary neurotrophic factor (CNTF) was a selective survival factor for rat VP-MCNs in organotypic cultures of the rat hypothalamic paraventricular nucleus (PVN) . We examined the effects of CNTF on the survival of these neurons in rat and mouse SONs . CNTF (10 ng/ml) in the culture media increased the survival of VP-MCNs by 6-fold and OT-MCNs by 3-fold . In the mouse, both OT- and VP-MCNs survive very well in organotypic cultures under standard culture conditions and the addition of CNTF had no further effect . Consistent with these results, in situ hybridization showed substantially higher levels of VP- and OT-mRNA in rat PVNs and SONs in the presence of CNTF, but produced no changes in these nuclei in the mouse . The optimum period for the survival effect of CNTF on MCNs in the rat hypothalamic cultures was in the first 7-10 days of culture and this effect is maintained for at least 5 additional days if CNTF is then removed from the medium . Therefore, using CNTF in the culture media can provide an opportunity for long-term studies of rat VP- and OT-MCNs in SONs in organotypic cultures .

J Infect Chemother, 2001 Dec, 7(4), 224 - 7
Distribution of Legionella longbeachae and other legionellae in Japanese potting soils; Koide M et al.; The distribution of Legionella longbeachae and Legionella spp . in Japanese potting soils was examined . Thirty samples were collected: 13 were composted wood products, 11 were potting mixes (containing composted wood products, sand, mineral fertilizer, and manure), 2 were peatmoss, 3 were peatmoss-sand mixes (containing peatmoss and sand), and 1 was a sample of hydroponic clay balls . A suspension of each sample was made in sterile distilled water and acidified, and 100 microl was plated on buffered charcoal yeast extract alpha (BCYEalpha) agar containing modified wadowsky yee (MWY) supplement and pimaricin (direct method) . In parallel, each suspension was incubated at 33 degrees C for several months to allow for amebic enrichment, if present; the suspensions were then plated onto culture media as described above (enrichment method) . A total of 46 strains of legionellae were isolated from 22 of the 24 samples (13 composted wood products, 11 potting mixes) . L . longbeachae was isolated from 9 samples . The most predominant species of legionellae in potting soils was Legionella bozemanii, which was isolated from 13 samples . Legionella spp . and L . micdadei were isolated from 8 and 7 samples each . Compared with findings in potting soils in Australia (26/45; 58%), Japanese potting soils had an only 8.3% (2/24) isolation rate for L . longbeachae by the direct method . The components of composted wood products were broadleaves such as oak and Japanese oak, in contrast to the pine and eucalypt used in Australia, which may account for the different isolation rates . However, the amebic enrichment method was useful in increasing the recovery of legionellae in potting soils . Legionellae were not isolated from the peatmoss samples, a result identical to findings in surveys of similar material in Europe.

J Am Soc Nephrol, 2002 Feb, 13(2), 394 - 9
Effects of PHEX antisense in human osteoblast cells; Shih NR et al.; X-linked hypophosphatemia (XLH) is an X-linked dominant disorder that is characterized by rachitic bone disease and hypophosphatemia due to renal phosphate transport defect . The candidate gene for XLH, PHEX, has recently been identified and found to share high homology with endopeptidases . PHEX is expressed in various tissues, including bones, and the available evidence today indicates that bones can release abnormal humoral factors that affect bone mineralization and proximal tubule phosphate transport in XLH . It was, therefore, hypothesized that the inactivating mutations of PHEX in bone may lead to the release of humoral factors and contribute to the phenotypic expression of the disease . To test this possibility, clones of MG-63 cells, a human osteoblast cell line, were produced and stably transfected with PHEX-antisense vectors, resulting in a decrease in PHEX expression at mRNA and protein levels . It was found that these antisense-transfected cells had impaired mineralization, with a decrease in 45Ca incorporation and calcification nodule formation . It was also found that the conditioned culture media collected from these antisense-transfected cells exhibited inhibitory activities on 45Ca incorporation by the nontransfected MG-63 cells and 32P uptake by the opossum kidney proximal tubular cells . The results of the study, therefore, provide strong evidence that supports the link between PHEX mutations and the pathogenesis of XLH.

Am J Respir Cell Mol Biol, 2002 Feb, 26(2), 209 - 15
Secreted and cell-associated adenylate kinase and nucleoside diphosphokinase contribute to extracellular nucleotide metabolism on human airway surfaces; Donaldson SH et al.; 5'-Nucleoside triphosphates (NTP) are present in the liquid covering airway surfaces and mediate important physiologic events through their interaction with P2-nucleotide receptors . Activation of airway P2Y(2) receptors, for example, stimulates ciliary beat frequency, chloride/liquid secretion, and goblet cell degranulation . We, therefore, have studied the metabolic pathways that regulate the concentration of nucleotides on airway surfaces . Stimulation of submucosal gland secretion in the nose was previously found to decrease the concentration of 5'-adenosine triphosphate (ATP) in nasal lavage samples due to the presence of a secreted 5'-nucleoside triphosphatase (NTPase) . In this study, gland secretions were further studied and found to also contain adenylate kinase (AK) and nucleoside diphosphokinase (NDPK) activities . Ecto-AK and ecto-NDPK activities were also detected in well-differentiated cultures of superficial nasal epithelia, which reflected a combination of cell-associated and released (into culture media) AK and NDPK activities . This study demonstrates that "ecto-kinases" on airway surfaces (1) emanate from different enzyme families, including both AK and NDPK; (2) are expressed at superficial epithelial surfaces and in submucosal glands; and (3) may be important regulators of nucleotide concentrations on airway surfaces.

J Vasc Surg, 2002 Jan, 35(1), 152 - 7
Validation of an in vitro model of human saphenous vein hyperplasia; Castronuovo JJ Jr et al.; OBJECTIVE: The purpose of this study was the validation of the physiologic appropriateness of in vitro organ culture of human saphenous vein as a model with the demonstration of the occurrence of the processes of cell proliferation, remodeling, and hyperplasia . METHODS: Saphenous vein from 28 patients was cross-sectioned into seven 2-mm segments and maintained in organ culture for 2 days or 2 weeks . Three organ culture media were used: a chemically well-defined medium (RPMI-1640) and the same medium supplemented with the undefined protein-containing supplements fetal bovine serum (FBS) or pooled adult human plasma (type AB) . The outcome measures at 2 days and 2 weeks were compared with measurements of segments from the same vein at the time of harvest . Excess saphenous vein harvested for arterial bypass grafting was obtained after approval of the study protocol by the Institutional Review Board . Cell proliferation was measured with immunostaining for proliferating cell nuclear antigen . Remodeling and intimal hyperplasia were measured with micromorphometric comparisons of vein segment cross-sectional area before and after organ culture . RESULTS: There was no evidence of cell death or tissue degeneration on histologic examination of the cultured vein segments . Cell proliferation, expressed as proliferation index (PI; positive proliferating cell nuclear antigen nuclei/total nuclei), significantly increased as compared with freshly harvested vein after 2 days of culture in undefined, protein-supplemented media (mean PI, 42.4 +/- 7.4%; P <.001) . A significant increase in cell proliferation did not occur in the defined, unsupplemented medium until 2 weeks (mean PI, 16.2 +/- 7.1%; P <.001) . The cross-sectional area of the vein wall increased during culture in all media . A statistically significant increase in the cross-sectional area of the vein wall occurred during culture with plasma (P <.001) and FBS supplementation (P =.002) . The increase in the cross-sectional area of the vein in defined media was almost statistically significant (P =.089) . A significant increase was seen in the cross-sectional area of the media (P =.006) and adventitia (P =.030) of veins cultured with plasma supplementation and in the cross-sectional area of the adventitia (P =.034) of veins cultured with FBS supplementation . CONCLUSION: These results show that human saphenous vein in culture is viable, shows cell proliferation, and exhibits remodeling of the layers of the vein wall . This is the first report to document hyperplasia in human vascular tissue cultured in a defined medium.

Trans Am Ophthalmol Soc, 2001, 99, 111 - 30; discussion 130-2
Curvularia keratitis; Wilhelmus KR et al.; PURPOSE: To determine the risk factors and clinical signs of Curvularia keratitis and to evaluate the management and outcome of this corneal phaeohyphomycosis . METHODS: We reviewed clinical and laboratory records from 1970 to 1999 to identify patients treated at our institution for culture-proven Curvularia keratitis . Descriptive statistics and regression models were used to identify variables associated with the length of antifungal therapy and with visual outcome . In vitro susceptibilities were compared to the clinical results obtained with topical natamycin . RESULTS: During the 30-year period, our laboratory isolated and identified Curvularia from 43 patients with keratitis, of whom 32 individuals were treated and followed up at our institute and whose data were analyzed . Trauma, usually with plants or dirt, was the risk factor in one half; and 69% occurred during the hot, humid summer months along the US Gulf Coast . Presenting signs varied from superficial, feathery infiltrates of the central cornea to suppurative ulceration of the peripheral cornea . A hypopyon was unusual, occurring in only 4 (12%) of the eyes but indicated a significantly (P = .01) increased risk of subsequent complications . The sensitivity of stained smears of corneal scrapings was 78% . Curvularia could be detected by a panfungal polymerase chain reaction . Fungi were detected on blood or chocolate agar at or before the time that growth occurred on Sabouraud agar or in brain-heart infusion in 83% of cases, although colonies appeared only on the fungal media from the remaining 4 sets of specimens . Curvularia was the third most prevalent filamentous fungus among our corneal isolates and the most common dematiaceous mold . Corneal isolates included C senegalensis, C lunata, C pallescens, and C prasadii . All tested isolates were inhibited by 4 micrograms/mL or less of natamycin . Topical natamycin was used for a median duration of 1 month, but a delay in diagnosis beyond 1 week doubled the average length of topical antifungal treatment (P = .005) . Visual acuity improved to 20/40 or better in 25 (78%) of the eyes . CONCLUSIONS: Curvularia keratitis typically presented as superficial feathery infiltration, rarely with visible pigmentation, that gradually became focally suppurative . Smears of corneal scrapings often disclosed hyphae, and culture media showed dematiaceous fungal growth within 1 week . Natamycin had excellent in vitro activity and led to clinical resolution with good vision in most patients with corneal curvulariosis . Complications requiring surgery were not common but included exophytic inflammatory fungal sequestration, treated by superficial lamellar keratectomy, and corneal perforation, managed by penetrating keratoplasty.

Bone, 2002 Jan, 30(1), 57 - 63
Rat costochondral chondrocytes produce 17beta-estradiol and regulate its production by 1alpha,25(OH)(2)D(3); Sylvia VL et al.; Prior studies have shown that 17beta-estradiol (17beta-E(2)) regulates growth plate chondrocyte maturation and differentiation . This study examines the hypothesis that 17beta-E(2) is a local regulator of rat costochondral growth plate chondrocytes by determining whether these cells express aromatase mRNA and enzyme activity, produce 17beta-E(2), and regulate 17beta-E(2) production by vitamin D(3) metabolites in a gender-specific and cell-maturation-dependent manner . Aromatase gene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis of total RNA from male and female chondrocytes . Aromatase specific activity was measured in cell layer lysates of confluent male and female rat costochondral resting zone (RC) and growth zone (GC) cartilage cells that had been treated for 24 h with 1alpha, 25(OH)(2)D(3), 24R,25(OH)(2)D(3), or transforming growth factor (TGF)-beta1 . 17beta-E(2) released into the culture media of treated cells was measured by radioimmunoassay (RIA) . Female RC cells expressed the highest levels of aromatase mRNA compared with male RC cells and both male and female GC cells . Aromatase activity was present in male and female cells and was 1.6 times greater in female RC cells than female GC cells; male RC and GC cells displayed comparable levels . All cultures produced 17beta-E(2), with a 2.5-fold greater production by female RC cells than female GC cells or either cell type from male rats . Treatment of cultures with 1alpha,25(OH)(2)D(3) caused a dose-dependent increase in 17beta-E(2) production by female RC (1.5-fold greater than control cells) and female GC (threefold greater than control cells) cells . In contrast, 1alpha,25(OH)(2)D(3) had no effect on male GC cells and increased production in male RC cells by only 10% at the highest concentration of 1alpha,25(OH)(2)D(3) used . Neither 24R, 25(OH)(2)D(3) nor TGF-beta1 had an effect on 17beta -E(2) production . These results support our hypothesis and indicate that 17beta-E(2) is most likely a local regulator of rat costochondral growth plate chondrocytes.

Anal Chem, 2001 Dec 15, 73(24), 6070 - 6
Detection of human immunodeficiency virus type 1 reverse transcriptase using aptamers as probes in affinity capillary electrophoresis; Pavski V et al.; An affinity capillary electrophoresis/laser-induced fluorescence (CE/LIF) assay was developed for direct and specific detection of reverse transcriptase (RI) of the type 1 human immunodeficiency virus (HIV-1) using fluorescently labeled single-stranded DNA aptamers as probes . The aptamer used (RT 26) is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT . An affinity complex of RT 26-HIV-1 RT was readily formed, and calibration curves were linear up to 50 nM (6 microg/mL) HIV-1 RT concentration, with both the free probe and complex peak usable for analytical quantitation . Cell culture media (RPMI with 10% fetal bovine serum) interfered with the assay and aptamer-HIV-1 RT binding . Nonspecific binding was observed in low or undiluted culture, necessitating at least 100-fold dilution for analysis of raw culture samples.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 139 - 44
An easy screening method for fungi producing ochratoxin A in pure culture; Bragulat MR et al.; A simple screening method has been developed for detecting ochratoxin production by fungi, based on high-performance liquid chromatographic determinations on extracts obtained from agar plugs cut from pure Petri dish cultures . Two culture media . Yeast Extract Sucrose agar and Czapek Yeast Extract agar, and three extraction solvents (methanol, methylene chloride/formic acid, and methanol/formic acid) were compared . All of the isolates tested produced ochratoxin A in one or both culture media after 7 or 14 days of incubation . Based on the results obtained, the use of both culture media is recommended . As extraction solvent, either methanol or methanol-formic acid could be used . This method also provides quantitative information on the level of ochratoxin produced by the cultures . The simplicity of the method makes it very useful when many fungal isolates need to be screened.

Cryo Letters, 2001 May-Jun, 22(3), 163 - 74
Effects of plant growth regulators on survival and recovery growth following cryopreservation; Turner SR et al.; Studies on the effects of plant growth regulators (PGRs) on survival, recovery and post-recovery growth of shoot apices following cryopreservation are limited . In this study, the effects of plant growth regulators in both the culture phase and the recovery phase of cryostorage were examined for the rare plant species, Anigozanthos viridis ssp terraspectans Hopper . Survival of shoot apices was not correlated to cytokinin or auxin treatments administered in culture media prior to cryostorage . In recovery media, the plant growth regulators, kinetin, zeatin (cytokinins), IAA, (auxin) and GA3 were examined for their effect following cryopreservation . It was found that the application of a combination of cytokinin and 0.5 microM GA3 from day zero was the most appropriate for obtaining vigorously growing plantlets following LN immersion . This combination proved to be more effective than basal medium, zeatin or kinetin treatments.

Adv Exp Med Biol, 2000, 483, 563 - 70
Cytoprotective effect of taurine against hypochlorous acid toxicity to PC12 cells; Kearns S et al.; Taurine has been shown to be an effective scavenger of hypochlorous acid (HOCl) . The role of HOCl is well established in tissue damage associated with reperfusion injury mediated by neutrophils . The role of HOCl in CNS injury and inflammatory reactions has not been well established . Myeloperoxidase activity is present in the CNS and it has been associated with ischemic injury . The aim of the present study was to determine the cytotoxicity of HOCl in a neuronal cell line (PC12) and the ability of taurine to prevent or reverse neurotoxicity . PC12 cells were grown in 96 well plates at a plating density of approximately 100,000 cells per well . HOCl was made up fresh from NaOCl for each experiment and the concentration verified spectrophotometrically . PC12 cells were exposed to HOCl for 1 hour in phosphate-buffered saline . Taurine was added at the time of HOCl treatment and in some experiments a post-treatment with taurine was performed by adding 1 or 10 mM taurine to the culture media (RPMI 1640) . The cells were allowed 24 hours to recover and viability was determined using a tetrazolium-based (MTT) assay . The first series of experiments evaluated the toxicity of HOCl and the efficacy of taurine to protect PC12 cells . HOCl at 50 microM reduced PC12 cell viability by 50% and 150 microM reduced viability to <25% of control levels . Taurine (0.5-20 mM) was tested for cytoprotection against 150 microM HOCl and PC12 cells treated with 0.5 mM taurine exhibited only a 20% reduction in viability compared to untreated controls . Taurine concentrations of 1 mM or higher provided nearly 100% protection against HOCl . A second study was performed comparing taurine to beta-alanine, glutathione and isethionic acid . HOCl (100 microM) reduced viability to 25 +/- 1% of controls and taurine, beta-alanine and glutathione at 1 mM provided nearly complete protection . In contrast, isethionic acid, which lacks an amino group, failed to provide protection . Taurine (1 or 10 mM) added after 50 microM HOCl treatment did not provide any protection and PC12 cell viability was reduced to <39% of controls . In contrast, if taurine (50 microM) was present during the HOCl treatment and 1 mM taurine was added after the treatment, PC12 cell viability was 80 +/- 5% of controls . A combination of 250 microM taurine during the HOCl treatment and 1 mM taurine post-treatment produced 100% protection . These results clearly show that taurine is an efficient scavenger of HOCl and can prevent neuronal damage caused by HOCl . Since myeloperoxidase expression in the CNS is increased by ischemia, one function of taurine released during an ischemic event may be to scavenge HOCl and provide neuroprotection.

J Reprod Fertil Suppl, 2001, 57, 423 - 33
The phenomenon and significance of teratospermia in felids; Pukazhenthi BS et al.; The common domestic cat is an important research model for endangered felids, as well as for studying genetic dysfunctions, infectious diseases and infertility in humans . Especially significant is the trait of teratospermia (ejaculation of < 40% morphologically normal spermatozoa) that commonly occurs in about 70% of the felid species or subspecies studied to date . Teratospermia, discovered more than two decades ago in the cheetah, is important: (i) for understanding the significance of sperm form and function; and (ii) because this condition is common in human males . It is apparent from IVF that deformed spermatozoa from teratospermic felids do not fertilize oocytes . However, the inability of spermatozoa from teratospermic males to bind, penetrate and decondense in the cytoplasm of the oocyte is not limited to malformed cells alone . Normal shaped spermatozoa from teratospermic males have reduced functional capacity . IVF results have consistently revealed a direct correlation between teratospermia and compromised sperm function across felid species and populations . The most significant differences between normospermic (> 60% normal spermatozoa per ejaculate) and teratospermic felids include: (i) the time required for sperm capacitation and the acrosome reaction to occur in vitro; (ii) culture media requirements for capacitation in vitro; (iii) phosphorylation patterns of tyrosine residues on sperm membrane proteins during capacitation; (iv) susceptibility to chilling-induced sperm membrane damage; (v) sensitivity to osmotic stress; (vi) stability of sperm DNA; (vii) sperm protamine composition; and (viii) fertilizing ability after intracytoplasmic sperm injection . In conclusion, (i) the felids (including wild species) are valuable for studying the functional significance of both pleiomorphic and normally formed spermatozoa from teratospermic donors, and (ii) the impact of teratospermia is expressed at both macrocellular and subcellular levels.

J Neurophysiol, 2002 Jan, 87(1), 640 - 4
Glycine receptors involved in synaptic transmission are selectively regulated by the cytoskeleton in mouse spinal neurons; van Zundert B et al.; Using whole cell patch-clamp recordings, we examined the effect of colchicine, a microtubule disrupter, on the properties of glycine receptors (GlyRs) in cultured spinal cord neurons . Confocal microscopy revealed that colchicine treatment effectively altered microtubule bundles and neuronal morphology . Application of colchicine via the culture media or the patch-pipette, however, did not affect the whole cell current rundown (73 +/- 6% of control after 1 h), the sensitivity of the GlyR to glycine (EC(50) = 29 +/- 1 microM), or strychnine inhibition (47 +/- 5% of control after 100 nM strychnine) . On the other hand, colchicine dialyzed for 25 min via the patch pipette selectively reduced the quantal amplitude of spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs) to 68 +/- 5% of control . This effect was specific for GlyRs since synaptic events mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABA(A) receptors were unchanged . In conclusion, this study indicates that microtubules can regulate the function of GlyRs involved in inhibitory synaptic transmission.

Eur J Biochem, 2002 Jan, 269(1), 337 - 46
Early growth response-1 gene (Egr-1) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro; Meyer RG et al.; The promoter of the early growth response gene (Egr-1) has been described to be activated by ionizing radiation, and it seems to be clear that this process involves different mitogen activated protein (MAP) kinases, dependent on the specific cell type examined . However, early steps leading to activation of the corresponding pathways and thus to overexpression of Egr-1 are not well understood . In this study, deletion mutants of the 5' upstream region of the Egr-1 gene were generated which allowed us to correlate the radiation-induction of the Egr-1 promoter in U87 glioma cells to five serum response elements . Based on the data shown, a possible role of two cAMP responsive elements for radiation-dependent promoter regulation could be ruled out . On the basis of activator/inhibitor studies applying fetal bovine serum, EGF, PD98059, anisomycin, SB203580, forskolin and wortmannin, it could be demonstrated that in U87 cells the ERK1/2 and potentially SAPK/JNK, but not the p38MAPK/SAPK2, pathway contribute to the radiation-induction of Egr-1 promoter . In addition, it was observed that irradiated cells secrete a diffusible factor into the culture media which accounts for the radiation-induced promoter upregulation . By blocking growth factor receptor activation with suramin, this effect could be completely abolished.

Zhonghua Fu Chan Ke Za Zhi, 2001 Feb, 36(2), 92 - 4
{Application of sequential culture in assisted reproductive technique}; Sun M et al.; OBJECTIVE: To investigate the effects of sequential culture technique on the outcomes of in vitro fertilization-embryo transfer(IVF-ET) . METHODS: Sequential culture media were used in 47 IVF-ET cycles, and conventional medium in 114 cycles . The fertilization rate, cleavage rate and speed, good embryo rate and pregnancy rate were compared between these 2 groups . RESULTS: There were significantly higher embryo cleavage and quality score rate, pregnancy rate (97.9%, 64.4%, 46.7%) in sequential media group as compared those in conventional medium group (89.7%, 40.2%, 28.2%) . CONCLUSION: Sequential culture system is more suitable for the development of cultured human oocytes and embryos.

Fertil Steril, 2002 Jan, 77(1), 114 - 8
Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome; Karaki RZ et al.; OBJECTIVE: To evaluate the efficacy of blastocyst culture and transfer in human in vitro fertilization (IVF) as compared to day 3 embryo transfer . DESIGN: Prospective randomized trial . SETTING: Private assisted reproduction unit . PATIENT(S): A total of 162 IVF patients were included in the day 3 embryo transfer (n = 82) and blastocyst transfer (n = 80) groups . INTERVENTION(S): Embryo transfer on day 3 after culture in the standard culture media and blastocyst transfer on day 5 or 6 after culture in the sequential culture media . MAIN OUTCOME MEASURE(S):Implantation and pregnancy rates, multiple gestation rate . RESULT(S): The implantation rate for embryos transferred at the blastocyst stage was significantly higher than that for embryos transferred on day 3 (26% vs . 13%) . The viable pregnancy rate was similar in both groups (29% vs . 26%) . Significantly fewer embryos were required for transfer at the blastocyst stage compared with day 3 embryo transfer (2.0 +/- 0.1 vs . 3.5 +/- 0.63) . The high-order multiple gestation rate was significantly less with the blastocyst transfer than with the day 3 embryo transfer (4% vs . 19%) . CONCLUSION(S): With the use of blastocyst culture, a few embryos can be transferred without decreasing the overall pregnancy rate . This may reduce multiple gestations and improve human IVF outcome.

In Vitro Cell Dev Biol Anim, 2001 Nov-Dec, 37(10), 656 - 67
Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers; Hamilton GA et al.; Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium) . Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk . For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures . Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content . Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism . Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation . For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal . These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed . Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture . Hepatocytes in spheroids formed bile canaliculi . and expressed an actin distribution resembling that found in hepatocytes in vivo . Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes . The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.

Theriogenology, 2002 Jan 1, 57(1), 256 - 73
In vitro production of embryos in swine; Abeydeera LR; In recent years, progress has been achieved in the production of pig embryos through IVM and IVF techniques . Cytoplasmic maturation of oocytes has been improved by modifications to IVM procedures . However, the historical problem of polyspermic penetration still remains a major issue to be solved . Recent studies indicate that the type of IVF medium and certain modifications to that medium can reduce polyspermy . Efforts should be directed to increase the developmental competence and quality of embryos . At present, many embryo culture (EC) media are available that can overcome the historical 4-cell block and support development of early in vivo derived embryos to the blastocyst stage . In contrast, blastocyst development of in vitro produced embryos in these culture media varies significantly . Furthermore, morphology and cell numbers in in vitro produced blastocysts are inferior to their in vivo counterparts . However, several modifications to EC techniques have improved embryo quality and developmental competence . Testing embryo viability through surgical transfer to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes . Although reliable in vitro systems are available for the generation of pig embryos, the problem of polyspermy and poor embryo development hamper their large-scale implementation . Further research efforts should be directed to improve oocyte/embryo quality and the methods to minimize polyspermy through development of novel IVM, IVF, and EC techniques.

Cell Mol Neurobiol, 2001 Aug, 21(4), 421 - 8
Establishment and partial characterization of a continuous human malignant glioma cell line: NG97; Grippo MC et al.; 1 . A human glioma cell line, NG97, was established from tissue obtained from a patient diagnosed with a grade III astrocytoma . 2 . The NG97 cell line has been subcultured for more than 100 passages in standard culture media without feeder layer or collagen coatings . 3 . NG97 cells grow in vitro as two subpopulations with distinct morphological appearance: stellate cells with pleomorphic nuclei, and small round cells with few processes . The cells have a doubling time of about 72 h and a plating efficiency of 1% . The injection of NG97 cells into congenitally athymic mice induced the formation of solid tumor masses that could be retransplanted every 4 weeks . The cells obtained from tumor mass when cultivated in vitro had a morphology comparable to those of the initial culture . 4 . This cell line may prove useful for cellular and molecular studies as well as in studies of gliomas treatment.

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