Culture medium

Korean J Ophthalmol, 1998 Dec, 12(2), 103 - 7
The ultrastructure of corneal epithelium after co-cultivation with herpes simplex virus; Lee DY et al.; To elucidate the ultrastructural change of corneal epithelium co-cultured with herpes simplex virus (HSV), the corneal epithelium of 3 rabbits was excised and cultivated in culture media . After 7 days, the Kos strain of herpes simplex virus was inoculated in the cultured cornea epithelium until cytopathic effect was occurred . It was fixed in the solution of 3% glutaraldehyde and examined with electronmicroscope . In co-cultured cells, the intercellular spaces had increased and microvilli were seen prominently . The virus particles that initiated the infection by fusing the viral envelope with the plasma membrane were also seen . The nuclear degeneration in an infected cell was prominent . The nuclear membrane was folded markedly, and the chromatin was degraded, condensed and displaced toward the nuclear membrane . Numerous viral particles and inclusion bodies were present in the nuclei . These findings suggest that the infectious process of herpes simplex virus in the human corneal epithelium may occur in a similar way . This result would be helpful in understanding the pathogenesis of herpes simplex epithelial keratitis.

Eur J Neurosci, 1999 Apr, 11(4), 1421 - 30
Cleavage of the TrkA neurotrophin receptor by multiple metalloproteases generates signalling-competent truncated forms; Diaz-Rodriguez E et al.; The ectodomain of the neurotrophin receptor TrkA has been recovered as a soluble fragment from the culture media of cells by a process that involves endoproteolytic cleavage . This cleavage may be upregulated by several treatments, including NGF treatment or protein kinase C activation . In this report we have investigated the cellular site and proteolytic activities involved in TrkA cleavage, and the effects of ectodomain truncation on signalling . Cleavage occurs when the receptor is at, or near, the cell surface, and it can be prevented by agents that affect protein sorting . Cleavage generates several cell-bound fragments, and their generation can be differentially blocked by inhibitors, documenting the involvement of multiple plasma membrane metalloendoproteases . The major cell-bound receptor fragment (i) is tyrosine-phosphorylated in vivo; (ii) does autophosphorylate in vitro; and (iii) is able to associate with intracellular signalling substrates . Artificial deletion of the TrkA ectodomain results in an active receptor that induced neurite outgrowth in pheochromocytoma cells . Cleavage by this natural cellular mechanism appears thus to serve not only as an outlet of receptor binding fragments, but also to generate signalling-competent cell-bound receptor fragments . In the nervous system this ligand-independent receptor activation could play important roles in the development and survival of neurons.

Am J Reprod Immunol, 1999 Feb, 41(2), 164 - 7
Interleukin-6 levels in co-culture of human in vitro fertilization embryos with Vero cells are not predictive of future successful development; Lapree-Delage G et al.; PROBLEM: In an attempt to predict successful embryo transfer and implantation, we measured interleukin (IL)-6 levels in culture supernatants of co-cultured preimplantation human embryos . We tested whether all in vitro fertilized human embryos in co-cultures do secrete IL-6, and whether there was any difference in such production between embryos that successfully reached the blastocyst stage and blocked embryos . We also addressed the question of IL-6 secretion by co-culture support cells, namely Vero cells themselves . METHOD OF STUDY: Each fertilized oocyte was cultured individually and transferred in culture wells supplemented with a feeder layer of Vero cells at day 2 . In vitro IL-6 production was measured by bioassay of the culture media . RESULTS: Because Vero cells themselves secrete IL-6, it became impossible, in co-culture, to quantify production of IL-6 by the sole embryos . On the other hand, the co-culture technique has shown us that embryos are likely to consume IL-6 . There was no difference between blastocysts and blocked embryos . CONCLUSIONS: IL-6 levels in human embryo co-cultures do not correlate with future successful embryo transfer.

J Androl, 1999 Jan-Feb, 20(1), 54 - 62
Hormonal regulation of inhibin B secretion by immature rat sertoli cells in vitro: possible use as a bioassay for estrogen detection; Depuydt CE et al.; The influences of follicle-stimulating hormone (FSH), gonadal steroids, and culture time were studied in relation to inhibin B production by Sertoli cells of immature rats cultured in vitro . Sertoli cell-enriched cultures were established from 18-day-old rats and were maintained in medium supplemented with insulin, transferrin, and epidermal growth factor at 34 degrees C . A recently developed ELISA for the measurement of inhibin B was used to assess the effects of recombinant human FSH (rh FSH), testosterone (T), and estradiol (E2) on inhibin B production and accumulation in the culture media of Sertoli cell-enriched cultures and to optimize the cell culture system to serve as a bioassay for the detection and quantification of estrogens and estrogenlike substances . Prolonging the incubation time (24, 48, or 72 hours) of Sertoli cells with control medium without rh FSH, T, or E2 resulted in a time-dependent increase of inhibin B production . Incubation with rh FSH (1, 2.5, 5, or 10 U/L) caused a dose- and time-dependent increase of inhibin B production by Sertoli cells (but not by cultured Leydig cells), reaching a plateau at 5 U/L rh FSH . Addition of T in concentrations of 2.88, 5, or 50 ng/ml to medium without rh FSH and E2 significantly lowered the daily production rate of inhibin B (P < 0.05) . In contrast, addition of E2 (0.01 and 0.1 ng/ml) caused a dose-responsive increase in inhibin B production after 24 and 48 hours . The relative increment of inhibin B production induced by E2 was maximal after 24 hours in the presence of 2.5 U/L rh FSH (acting synergistically) and in the absence of T . When these conditions are implemented, the Sertoli cell culture system may serve as a bioassay for estrogenic substances, and it may reflect the possibly harmful effect they may have on spermatogenesis.

Hum Reprod, 1999 Feb, 14(2), 395 - 9
Induction of macrophage migration inhibitory factor in human ovary by human chorionic gonadotrophin; Wada S et al.; The role of macrophage migration inhibitory factor (MIF) in human ovarian function remains obscure . The aim of this study was to investigate how MIF was related to ovulation by quantitative analysis of serum, follicular fluid and culture medium of granulosa cells obtained from in-vitro fertilization (IVF) and embryo transfer patients . Serum MIF concentrations in ovarian stimulation cycles for IVF-embryo transfer were higher at day 1 (median 92.6 ng/ml), which took place 35 h after human chorionic gonadotrophin (HCG) administration and just before the retrieval of oocytes, than those before day -6 (12.1 ng/ml), at day -5 to about day 0 (17.5 ng/ml) or at day 2 to about day 14 (8.2 ng/ml) . MIF concentrations in the follicular fluid (113.4 ng/ml) obtained in ovarian stimulation cycles for IVF-embryo transfer were significantly higher than in serum (72.0 ng/ml) collected at the same time . MIF concentrations in the follicular fluid in natural cycles were higher in the ovulatory phase (51.6 ng/ml) than in the late follicular phase (13.8 ng/ml) . MIF concentrations in the culture media of granulosa cells increased from 3.2 ng/ml to 7.2 ng/ml with HCG stimulation, and decreased from 2.4 ng/ml to 1.2 ng/ml when stimulation was withheld . These results indicate that HCG can induce the elevation of serum and follicular fluid MIF concentrations through the stimulation of ovarian cells, and that MIF is probably involved in the mechanism of ovulation.

Cell Mol Biol (Noisy-le-grand), 1999 Feb, 45(1), 137 - 52
Expression of Glc3Man9GlcNAc2-PP-Dol is a prerequisite for capillary endothelial cell proliferation; Martinez JA et al.; Protein N-glycosylation has been proposed to be intimately involved in the migration, proliferation and differentiation of endothelial cells . Using a synchronized, non-transformed capillary endothelial cell line from bovine adrenal medulla as a model, and the N-glycosylation inhibitor, tunicamycin, we have elucidated the molecular basis of the dolichol pathway in the angiogenic process . The synchronized culture required approximately 68 hrs . to complete one cell cycle, cells spending nearly 36 hrs . in G1 phase, 8 hrs . in S phase and 24 hrs . in G2 + M phase when maintained in 2% fetal bovine serum (heat-inactivated) . The cell cycle however, was shortened due to a reduction of the G1 phase by 12-16 hrs . when the serum concentration was increased to 10%, or when beta FGF (1 or 10 nanogram) was added into the culture media containing 2% serum . Light microscopy and scanning electron microscopy both supported these proliferative responses . Serum concentration below 2% arrested cell proliferation and induced capillary lumen-like structure formation with 48 hrs . Expression of the blood clotting antigen factor VIII:C (a M(r) 270,000 dalton N-linked glycoprotein and a marker of our endothelial cells) preceded the endothelial cell proliferation and established a temporal relationship . Tunicamycin, an inhibitor of Glc3Man9GlcNAc2-PP-Dol biosynthesis, a prerequisite for N-linked protein glycosylation in the ER-inhibited the cell growth and proliferation in a time and dose-dependent manner with a concomitant accumulation of immunopositive, non-glycosylated factor VIII:C in the conditioned media . Tunicamycin also caused surface blebbing and induction of programmed cell death (PCD)(apoptosis) within 32 hrs . Absence of cellular growth and proliferation, surface blebbing and the induction of PCD in the presence of tunicamycin, provided conclusive evidence that normal expression of Glc3Man9GlcNAc2-PP-Dol is an essential event for capillary proliferation during angiogenesis.

Osaka City Med J, 1998 Dec, 44(2), 195 - 200
Alteration in the differentiated phenotypes of cultured chondrocytes from human articular cartilage under various culture conditions; Yutani Y et al.; Chondrocytes produce proteoglycans and type II collagen as their main matrix components, which can also be considered to be indices of their differentiated phenotype . Each differentiation step occurs in accordance with the environment of each cell . The analysis of the properties of chondrocytes has mostly been performed in cultured cells . In this report, we examined the effects of the culture media and fetal calf serum on cultured human articular chondrocytes . It was confirmed that the quantity of proteoglycan produced by the chondrocytes changed according to the different kinds of media used, and that the molecular size of the proteoglycans showed a tendency towards de-differentiation with decreasing fetal calf serum concentrations . We conclude that our experimental results which are obtained in cultured human articular chondrocytes should be discussed with consideration to the finding of this report.

J Mol Cell Cardiol, 1999 Feb, 31(2), 377 - 86
Hypoxia-reoxygenation and polyunsaturated fatty acids modulate adrenergic functions in cultured cardiomyocytes; Delerive P et al.; The polyunsaturated fatty acids (PUFAs) of the omega 3 series are known to modulate adrenergic functions in ventricular myocytes . This study evaluated the influence of hypoxia duration and PUFA composition on the ability of cultured rat cardiomyocytes in producing alpha- and beta-adrenergic messengers (IPs and cAMP) . After hypoxia (1.5, 2.5 or 3.5 h) followed by reoxygenation (1h) . IP and cAMP production was induced by phenylephrine or isoproterenol stimulation, respectively . Hypoxia did not affect the basal level of messenger production in unstimulated cells, but decreased the cAMP production elicited by isoproterenol stimulation (up to 50%) . The decrease in IP production after phenylephrine stimulation was observed only after long-term hypoxia duration close to irreversible cellular damages . The use of modified culture media supplemented with either arachidonic acid (AA) or docosahexaenoic acid (DHA) induced cardiomyocytes displaying either an arachidonic acid membrane profile (35% AA and 2% DHA in the phospholipids) or a docosahexaenoic acid membrane profile (15% AA and 20% DHA) . These modifications did not alter the basal level of either messenger production in unstimulated cells nor the IP released after alpha-adrenergic stimulation . Conversely, the decrease in cAMP production was significantly more pronounced in docosahexaenoic acid-enriched cells than in arachidonic acid-enriched cells . This study suggests that hypoxia alters the beta-adrenergic messenger production, and that the alpha-system may balance the depression of the beta-system . The depression of the beta-adrenergic function induced by the incorporation of docosahexaenoic acid in membrane phospholipids may contribute to the beneficial effect of this fatty acid in the reperfused heart.

Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 74 - 8
Bystander macrophages silence transgene expression driven by the retroviral long terminal repeat; Kitamura M; The Moloney murine leukemia virus (MLV)-based retroviral vector has been widely used for transfer of exogenous genes to various organs and tissues . Although the long terminal repeat (LTR) of MLV allows for transgene expression in a wide range of cell type, its activity is often silenced in vivo . In reporter macrophages transduced with a MLV-based retroviral vector, activity of the LTR was transiently and reversibly suppressed following stimulation by lipopolysaccharide (LPS) . When unstimulated reporter macrophages were co-cultured with LPS-stimulated, untransduced macrophages, the LTR activity was similarly depressed . Activity of the LTR in retrovirus-transduced, mesangial cells was also down-regulated when co-cultured with activated macrophages . This suppressive effect was reproduced by cross-feeding with culture media conditioned by activated macrophages . LPS-stimulated macrophages abundantly expressed cytokines including IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) . When externally added, TNF-alpha and/or TGF-beta1, but not IL-1beta, depressed activity of the LTR in reporter macrophages and reporter mesangial cells . These results raise a possibility that expression of transgenes driven by the MLV-LTR may be silenced in vivo when the retrovirally-transduced cells are co-localized with activated macrophages .

J Cell Physiol, 1998 Dec, 177(4), 636 - 45
Isolation and characterization of a cDNA clone encoding a novel peptide (OSF) that enhances osteoclast formation and bone resorption; Reddy S et al.; Using an expression cloning approach, we identified and cloned a novel intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption, termed OSF . Conditioned media from 293 cells transiently transfected with the 0.9 kb OSF cDNA clone stimulated osteoclast-like cell formation in both human and murine marrow cultures in the presence or absence 10(-9) M 1,25-dihydroxyvitamin D3 . In addition, conditioned media from 293 cells transfected with the OSF cDNA clone enhanced the stimulatory effects of 1,25-(OH)2D3 on bone resorption in the fetal rat long bone assay . In situ hybridization studies using antisense oligomers showed expression of OSF mRNA in highly purified osteoclast-like cells from human giant cell tumors of the bone . Northern blot analysis demonstrated ubiquitous expression of a 1.3 kb mRNA that encodes OSF in multiple human tissues . Sequence analysis showed the OSF cDNA encoded a 28 kD peptide that contains a c-Src homology 3 domain (SH3) and ankyrin repeats, suggesting that it was not a secreted protein, but that it was potentially involved in cell signaling . Consistent with these data, immunoblot analysis using rabbit antisera against recombinant OSF demonstrated OSF expression in cell lysates but not in the culture media . Furthermore, recombinant OSF had a high affinity for c-Src, an important regulator of osteoclast activity . Taken together, these data suggest that OSF is a novel intracellular protein that indirectly enhances osteoclast formation and osteoclastic bone resorption through the cellular signal transduction cascade, possibly through its interactions with c-Src or other Src-related proteins.

Hum Reprod, 1998 Dec, 13 Suppl 4, 218 - 25
Development of serum-free media for the culture and transfer of human blastocysts; Gardner DK; The culture and transfer of the blastocyst stage embryo has several advantages for assisted reproduction in the human . However, due to inadequacies of present culture conditions in human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development around the 4- to 8-cell stage, with resultant implantation rates of only 10-25% . In other mammalian species the transfer of cleavage stage embryos, which normally reside in the oviduct, results in a significantly lower implantation rate compared with the transfer of blastocysts . Extended culture of human embryos in vitro will help to identify those embryos with little, if any, developmental potential . It is therefore plausible that the blastocyst has an intrinsically higher viability than the cleavage stage embryo . It has now been shown in human IVF that sequential serum-free media can support > 50% blastocyst development, with an implantation rate per blastocysts of 50%, double that obtained for cleavage stage embryos . As the implantation rate of the blastocyst is higher than the cleavage stage embryo, fewer blastocysts are required for transfer . The development of completely defined embryo culture media may prove feasible by the replacement of protein with the glycosaminoglycan hyaluronate . Hyaluronate, which is protein-free, is more suitable than albumin in supporting implantation in the mouse, and can eliminate the biological variation inherent when using protein and the potential for contamination when using blood products such as albumin.

Hum Reprod, 1998 Dec, 13 Suppl 4, 173 - 83
Is the mouse a good model for the human with respect to the development of the preimplantation embryo in vitro?
Quinn P, Horstman FC.
A comparison has been made of various aspects of preimplantation development of mouse and human embryos in vitro . Changes in substrate utilization follow similar patterns in both species . This similarity in metabolic parameters between the two species has facilitated the use of the mouse as a model to study the formulation of culture media to be used at different stages over the preimplantation period from fertilization to the fully expanded blastocyst stage . It has also prescribed the mouse embryo as a practical tool for quality control testing of the laboratory system in human in-vitro fertilization . Aspects of the physiology of both species that require further study are the physiological levels of endogenous inorganic phosphate in the female reproductive tract, the requirement for inorganic phosphate in culture medium, the specificity of the amino acid requirements for optimal development before and after compaction and the importance of including EDTA in culture medium.

Hum Reprod, 1998 Dec, 13 Suppl 4, 146 - 55
The origin, effects and control of air pollution in laboratories used for human embryo culture; Hall J et al.; Testing shows that most laboratories conducting human gamete and embryo culture have air quality and sources of contamination that exceed the levels measured in homes, businesses and schools . The sources of these contaminants have been shown to be either from activities outside the laboratory, or emitted from materials used in the facility, such as compressed gas, cleaning and sterilizing agents, plastic and stored materials . Both the laboratory structure and the air handling systems may affect the air composition . The significance of these findings is being validated by the accumulation of field case studies and now by assay procedures . Products given off by road sealant were shown to have accumulated in one of the examined laboratories, adjacent to a large re-surfaced parking area . Aldehydes such as acrolein, hexanal, decanal, pentanal and others were detected at elevated concentrations that were statistically significant . Since it is not appropriate to add potentially suspect chemicals to human embryos, we used a mouse-model to study the effect of acrolein . The growth of mouse embryos was significantly affected after acrolein was added at different concentrations to the culture environment . The physiological effect was noted at concentrations in the low ppm range . The testing end-point of embryo death must still be considered to be a crude basis for evaluating toxicological effects, since it involves addition of compounds to culture media and unprotected growth until the blastocyst stage . The findings may, however, support observations of decreased pregnancy rate following exposure of human embryos to aldehydes or other adverse conditions . With proper engineering and material selection, it is possible to reduce such contamination . The usefulness of this approach for controlling aldehydes has been demonstrated by decreasing levels in the laboratory to below those of the outside air.

Early Pregnancy, 1997 Dec, 3(4), 291 - 300
The relationship between trophoblast differentiation and the production of bioactive hCG; Ho HH et al.; We previously showed that a significant number of failing pregnancies are associated with production of human chorionic gonadotropin (hCG) having relatively low bioactivity . The present study was designed to compare the secretion of intact, immunoreactive hCG to the secretion of bioactive hCG during trophoblast differentiation, and to test the hypothesis that the lower bioactive: immunoreactive hCG ratios in failing pregnancies are related to reduced or impaired trophoblast differentiation . Cytotrophoblast cells were isolated from term placentas and cultured under conditions that induced or did not induce syncytiotrophoblast formation . Culture media were collected at regular intervals up to 72 h and levels of immunoreactive and bioactive hCG were measured . The differentiation of cytotrophoblast cells to multinucleated syncytiotrophoblast was monitored by immunocytochemistry and electron microscopy . During the 72 h culture period, concentrations of immunoreactive and bioactive hCG increased in both differentiating and non-differentiating cells . However, the concentrations of immunoreactive and bioactive hCG were higher under culture conditions that promoted trophoblast differentiation . Furthermore, the ratio of bioactive hCG to immunoreactive hCG was higher in differentiating cultures . When differentiation was inhibited by dimethyl sulfoxide, the secretion of bioactive hCG was reduced and the bioactive: immunoreactive hCG ratio did not change . These findings are consistent with the idea that production of bioactive hCG accompanies syncytiotrophoblast formation.

Early Pregnancy, 1997 Sep, 3(3), 190 - 8
Expression and production of interleukin-10 by human trophoblast: relationship to pregnancy immunotolerance; Bennett WA et al.; Interleukin-10 (IL-10) is a T-helper type-2 (Th2) cytokine noted for its ability to suppress cytokine synthesis by T-helper type-1 (Th1) cells . IL-10 may play a role in pregnancy immunotolerance through the establishment of a Th2 cytokine bias at the maternal-fetal interface . This study examines the expression and production of IL-10 by normal and malignant human trophoblast . Term placental biopsies, cloned choriocarcinoma cell lines and isolated human trophoblast were utilized for the study of IL-10 expression . Choriocarcinoma cells (BeWo, JEG-3, JAR) were maintained in T-flask culture until confluence and then harvested by enzymatic dispersion . Purified term trophoblast were obtained by sequential trypsin/DNAse digests and CD9 immunoaffinity chromatography . Amplified IL-10 mRNA was detected by a reverse transcriptase polymerase chain reaction (RTPCR) technique . BeWo cells were maintained in artificial capillary culture (ACC) and conditioned media assayed for IL-10 . Granulocyte macrophage-colony stimulating factor (GM-CSF; 1.0, 10.0 and 100.0 ng/ml) was added to the BeWo cultures to examine its effects on trophoblast IL-10 production . IL-10 determinations were performed using a human ELISA system . IL-10 mRNA was detected in each trophoblast cell type examined with the exception of the JEG-3 choriocarcinoma cell line . IL-10 protein was also detected (range 6-22 pg/ml) in BeWo media on days 8 to 11 of culture . When serum was reduced in the culture media, IL-10 levels fell below the sensitivity of the assay (5 pg/ml) . Subsequent addition of GM-CSF stimulated BeWo IL-10 secretion in a dose-related fashion . These results support the concept IL-10 is expressed at the human maternal-fetal interface, and production of this important immunoregulatory molecule may be regulated, in part, by GM-CSF.

Biol Reprod, 1999 Apr, 60(4), 821 - 7
Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate; Mitalipov SM et al.; The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos . Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period . The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively) . Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied . Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium . In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete . A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes . Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage . When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused nuclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocyst stage.

Jpn J Pharmacol, 1999 Jan, 79(1), 33 - 40
Hydrogen peroxide-induced apoptosis and necrosis in human lung fibroblasts: protective roles of glutathione; Teramoto S et al.; Although reactive oxygen species (ROS)-related cell damage has been implicated in pathogenesis of fibrogenetic pulmonary disorders, features of ROS-mediated cell death in human lung fibroblasts are not completely understood . We therefore examined the effects of hydrogen peroxide (H2O2) on cell growth kinetics in human lung fibroblasts (HFL-1 cells) and tested the roles of antioxidants on the H2O2-induced cell death (i.e., necrosis and apoptosis) in HFL-1 cells . We found that the relatively low concentrations of H2O2 ranging from 10 microM to 100 microM induced predominantly apoptosis, whereas higher concentration of H2O2 ranging 1 mM-10 mM induced predominantly necrosis in HFL-1 cells . Extracellular supplementation of glutathione (GSH) in culture media significantly abolished the H2O2-induced cell death, whereas GSH-depleted cells by pretreatment with buthionine sulfoxime (BSO) were likely to undergo cell death caused by a lower concentration of H2O2 than normal HFL-1 cells without BSO treatment . These results indicate that H2O2 induces both necrosis and apoptosis of human lung fibroblasts at least in part through the action of ROS and that modulation of the ROS production inside and outside of cells may influence the cell survival during oxidative insults.

Curr Eye Res, 1999 Jan, 18(1), 62 - 71
TGF-beta elicits fibronectin secretion and proliferation in cultured chick lens epithelial cells; Richiert DM et al.; PURPOSE: To determine if the cataract forming influence of TGF-beta on lens cells is due to its effects on the ECM . METHODS: Primary cultures of chick lens annular pad cells were exposed to TGF-beta and various exogenously supplied components of the lens capsule . Proliferative response were measured through tritiated thymidine incorporation into DNA . Cell spreading accompanying increased matrix interactions and growth was monitored with phase contrast microscopy . ECM proteins were detected in culture media and as deposited matrices with Western blotting and silver staining . TGF-beta receptors were identified with Western blotting . RESULTS: Chick lens cells were shown to express type I and II TGF-beta receptors . TGF-beta stimulated cell growth and ECM production particularly with regard to fibronectin . Fibronectin was secreted into the culture medium and deposited onto plastic substrates . Plating cells on ECM components found in the lens capsule further increased their growth in response to TGF-beta . CONCLUSIONS: These results indicate that TGF-beta may have a normal function in the lens regulating capsular protein production . The potent stimulation of lens cell growth by TGF-beta may be due to mis-regulated production of lens capsular proteins not normally found in great abundance.

J Gen Virol, 1999 Feb, 80 ( Pt 2), 437 - 40
The R27080 glycoprotein is abundantly secreted from human cytomegalovirus-infected fibroblasts; Mullberg J et al.; A 45 kDa glycoprotein was purified from the culture media of human cytomegalovirus (HCMV)-infected fibroblasts . N-terminal sequencing revealed that the protein, R27080, is the translation product of the R27080 open reading frame of HCMV . R27080 is highly glycosylated and contains no cysteine or methionine residues . Proteolytic cleavage of R27080 by a furin-like enzyme was analysed in transfected COS-7 cells . R27080 is the first identified viral protein secreted from HCMV-infected cells.

Alcohol Clin Exp Res, 1999 Feb, 23(2), 363 - 70
Chronic ethanol upregulates NMDA and AMPA, but not kainate receptor subunit proteins in rat primary cortical cultures; Chandler LJ et al.; The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine . Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12 . Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course . Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13 . However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95 . In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine . These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins . Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.

Blood, 1999 Mar 15, 93(6), 1895 - 905
Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine alpha-L-iduronidase deficiency; Lutzko C et al.; Canine alpha-L-iduronidase (alpha-ID) deficiency, a model of the human storage disorder mucopolysaccharidosis type I (MPS I), is an ideal system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells . We performed adoptive transfer of genetically corrected autologous hematopoietic cells in dogs with alpha-ID deficiency . Large volume marrow collections were performed on five alpha-ID-deficient dogs . Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks of culture to retroviral vectors bearing the normal canine alpha-ID cDNA . Transduced LTMC cells from deficient dogs expressed enzymatically active alpha-ID at 10 to 200 times the levels seen in normal dogs . An average of 32% of LTMC-derived clonogenic hematopoietic cells were provirus positive by polymerase chain reaction and about half of these expressed alpha-ID . Approximately 10(7) autologous gene-modified LTMC cells/kg were infused into nonmyeloablated recipients . Proviral DNA was detected in up to 10% of individual marrow-derived hematopoietic colonies and in 0.01% to 1% of blood and marrow leukocytes at up to 2 to 3 years postinfusion . Despite good evidence for engraftment of provirally marked cells, neither alpha-ID enzyme nor alpha-ID transcripts were detected in any dog . We evaluated immune responses against alpha-ID and transduced cells . Humoral responses to alpha-ID and serum components of the culture media (fetal bovine and horse sera and bovine serum albumin) were identified by enzyme-linked immunosorbent assay . Cellular immune responses to autologous alpha-ID but not neo(r) transduced cells were demonstrated by lymphocyte proliferation assays . To abrogate potential immune phenomena, four affected dogs received posttransplant cyclosporine A . Whereas immune responses were dampened in these dogs, alpha-ID activity remained undetectable . In none of the dogs engrafted with genetically corrected cells was there evidence for clinical improvement . Our data suggest that, whereas the alpha-ID cDNA may be transferred and maintained in approximately 5% of hematopoietic progenitors, the potential of this approach appears limited by the levels of provirally derived enzyme that are expressed in vivo and by the host's response to cultured and transduced hematopoietic cells expressing foreign proteins.

Exp Anim, 1999 Jan, 48(1), 59 - 62
Production of germfree mice by embryo transfer; Okamoto M et al.; We applied the embryo transfer technique to germfree (GF) mouse production . Embryos harvested from superovulated mice were transferred aseptically, in a sterile environment, to the uterus of GF recipient females which had been mated with vasectomized GF males . One of the recipients became pregnant and delivered offspring . Sterility tests confirmed that the vasectomized males, newborns, recipient female mice, embryo-containing culture media, and the inside of the vinyl film isolator were germfree . These results suggest that the embryo transfer technique can be successfully applied to the production of GF mice.

Circ Res, 1999 Mar 5, 84(4), 458 - 66
Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes; Aikawa R et al.; -Mechanical stress induces a variety of hypertrophic responses, such as activation of protein kinases, reprogramming of gene expression, and an increase in protein synthesis . In the present study, to elucidate how mechanical stress induces such events, we examined the role of Rho family small GTP-binding proteins (G proteins) in mechanical stress-induced cardiac hypertrophy . Treatment of neonatal rat cardiomyocytes with the C3 exoenzyme, which abrogates Rho functions, suppressed stretch-induced activation of extracellular signal-regulated protein kinases (ERKs) . Overexpression of the Rho GDP dissociation inhibitor (Rho-GDI), dominant-negative mutants of RhoA (DNRhoA), or DNRac1 significantly inhibited stretch-induced activation of transfected ERK2 . Overexpression of constitutively active mutants of RhoA slightly activated ERK2 in cardiac myocytes . Overexpression of C-terminal Src kinase, which inhibits functions of the Src family of tyrosine kinases, or overexpression of DNRas had no effect on stretch-induced activation of transfected ERK2 . The promoter activity of skeletal alpha-actin and c-fos genes was increased by stretch, and these increases were completely inhibited by either cotransfection of Rho-GDI or pretreatment with C3 exoenzyme . Mechanical stretch increased phenylalanine incorporation into cardiac myocytes by approximately 1.5-fold compared with control, and this increase was also significantly suppressed by pretreatment with C3 exoenzyme . Overexpression of Rho-GDI or DNRhoA did not affect angiotensin II-induced activation of ERK . ERKs were activated by culture media conditioned by stretch of cardiomyocytes without any treatment, but not of cardiomyocytes with pretreatment by C3 exoenzyme . These results suggest that the Rho family of small G proteins plays critical roles in mechanical stress-induced hypertrophic responses.

Brain Res, 1999 Mar 13, 821(2), 530 - 4
Lithium decreases Cl--ATPase activity and increases intracellular Cl- concentration in cultured rat hippocampal neurons; Yagyu K et al.; Under the conditions of stimulated phosphatidylinositol turnover (0 . 1 mM carbachol plus 20 mM KCl), LiCl (0.1-10 mM) reduced the activity of Cl--ATPase in cultured rat hippocampal neurons without affecting Na+/K+- or anion-insensitive Mg2+-ATPase . This inhibition of Cl--ATPase was attenuated by the addition of 0.5 mM inositol to culture media . The intracellular Cl- concentrations of the LiCl-treated neurons increased in an inositol-sensitive manner .

Clin Exp Allergy, 1999 Jan, 29(1), 52 - 9
Diesel exhaust particulates upregulate histamine receptor mRNA and increase histamine-induced IL-8 and GM-CSF production in nasal epithelial cells and endothelial cells; Terada N et al.; BACKGROUND: Histamine is the most important chemical mediator in the pathogenesis of nasal allergy . Diesel exhaust particulates (DEPs) are common air pollutants from diesel engine-powered car exhaust and cause chronic airway diseases . Recently we observed that the nasal reactivity to histamine was enhanced in diesel exhaust-exposed guinea-pigs . It was also revealed that epithelial cells and endothelial cells in the airway produce certain cytokines in response to histamine . OBJECTIVE: We examined the effects of DEP extract on the expression of histamine H1 receptor (H1R) mRNA in human nasal epithelial cells (HNECs) and human mucosal microvascular endothelial cells (HMMECs), and on the production of IL-8 and GM-CSF induced by histamine . METHODS: HNECs and HMMECs were isolated from human nasal mucosa specimens . HNEC and HMMEC monolayers were cultured in the presence or absence of DEP extract . The change in the expression of H1R mRNA was then evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and the Southern blot analysis . To investigate the effects of DEP extract on the histamine-induced cytokine production, HNEC and HMMEC monolayers were cultured in the presence or absence of DEP extract for 3-24 h . After three washes with PBS, they were then incubated with 10(-6) mol/L histamine for 24 h . The amounts of IL-8 and GM-CSF in the culture media were measured by enzyme-linked immunoabsorbent assay . RESULTS: DEP extract increased the expression of H1R mRNA in both HNECs and HMMECs . The amount of IL-8 and GM-CSF, induced by histamine, was significantly higher in DEP extract pretreated HNECs and HMMECs than nontreated HNECs and HMMECs . CONCLUSION: These results strongly suggest that DEP accelerates the inflammatory change by not only directly upregulating H1R expression but also increasing histamine-induced IL-8 and GM-CSF production.

Biol Trace Elem Res, 1998 Winter, 66(1-3), 237 - 59
Adverse reproductive and developmental effects in Xenopus from insufficient boron; Fort DJ et al.; Frog embryo teratogenesis assay--Xenopus (FETAX) was utilized as a model system to evaluate the effects on embryo-larval development at various low boron (B) exposure levels in the culture media . Concentrations tested ranged from < 1 to 5000 microg B/L . A statistically significant (P < 0.05) increase in malformations was observed at < or = 3 microg B/L, but not at the greater concentrations . Abnormal development of the gut, craniofacial region and eye, visceral edema, and kinking of the tail musculature (abnormal myotome development) and notochord were observed . In subsequent studies, adult frogs were maintained for 28 d on two diets: (1) low B (LB, 62 microg B/kg) or (2) boric acid supplemented (BA, 1851 microg B/kg); the frogs were subsequently mated, and their offspring were cultured in media containing various levels of B . Results of the 28-d depletion studies indicated that frogs maintained under LB conditions produced a greater proportion of (1) necrotic eggs and (2) fertilized embryos, which abnormally gastrulated at a greater rate and were substantially less viable than embryos from frogs fed the BA diet . Malformations similar to those seen in the initial study were observed in embryos from the B-depleted adults maintained in an LB environment; 28 d on the LB diet enhanced the incidence of malformations associated with the LB culture media . These abnormalities were not observed in embryos cultured in > or = 4 microg B/L from adults cultured on the BA diet . These studies showed that insufficient B reproducibly interfered with normal Xenopus laevis development during organogenesis, substantially impaired normal reproductive function in adult frogs, and thus represent the first studies demonstrating the nutritional essentiality of B in an amphibian species.

Wien Klin Wochenschr, 1998 Dec 23, 110(24), 863 - 5
Growth of infectious and non-infectious B . burgdorferi at different salt concentrations; Elias A et al.; Borrelia burgdorferi, the causative agent of Lyme disease, grows in vitro in modified Barbour-Stoenner-Kelly (BSK-H) medium . We have studied the effect of increased osmotic strength of culture media on growth of infectious and non-infectious B . burgdorferi strains B31 and N40 . Relatively small increases in the NaCl concentration of the medium significantly inhibited growth in infectious as well as non-infectious strains . Growth of low passage, infectious clone B31-4a was more sensitive to increased NaCl concentrations than high passage, non-infectious clone B31-a . Growth of two infectious N40 strains, one low passage (N40-Lp) and one high passage (N40-P31) was more resistant to increased NaCl concentration than growth of infectious B31-4a . Osmotic strength is an important physical parameter for growth of B . burgdorferi in vitro and could influence its ability to adapt and to establish an infection within ticks and mammals.

J Neurochem, 1999 Mar, 72(3), 1139 - 45
Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice; Pelled D et al.; Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) . The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown . In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice . The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells . The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine . In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides . Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.

Toxicol Appl Pharmacol, 1999 Feb 15, 155(1), 24 - 31
Inhibition by lead of production and secretion of transthyretin in the choroid plexus: its relation to thyroxine transport at blood-CSF barrier; Zheng W et al.; Long-term, low-dose Pb exposure in rats is associated with a significant decrease in transthyretin (TTR) concentrations in the CSF . Since CSF TTR, a primary carrier of thyroxine in brain, is produced and secreted by the choroid plexus, in vitro studies were conducted to test whether Pb exposure interferes with TTR production and/or secretion by the choroid plexus, leading to an impaired thyroxine transport at the blood-CSF barrier . Newly synthesized TTR molecules in the cultured choroidal epithelial cells were pulse-labeled with {35S}methionine . {35S}TTR in the cell lysates and culture media was immunoprecipitated and separated by SDS-PAGE, and quantitated by autoradiography and liquid scintillation counting . Pb treatment did not significantly alter the protein concentrations in the culture, but inhibited the synthesis of total {35S}TTR (cells + media), particularly during the later chase phase . Two-way ANOVA of the chase phase revealed that Pb exposure (30 microM) significantly suppressed the rate of secretion of {35S}TTR compared to the controls (p < 0.05) . Accordingly, Pb treatment caused a retention of {35S}TTR by the cells . In a two-chamber transport system with a monolayer of epithelial barrier, Pb exposure (30 microM) reduced the initial release rate constant (kr) of {125I}T4 from the cell monolayer to the culture media and impeded the transepithelial transport of {125I}T4 from the basal to apical side of epithelial cells by 27% . Taken together, these in vitro data suggest that sequestration of Pb in the choroid plexus hinders the production and secretion of TTR by this tissue . Consequently, this may alter the transport of thyroxine across this blood-CSF barrier .

Alcohol Clin Exp Res, 1999 Jan, 23(1), 46 - 51
Effects of ethanol on alpha-adrenergic and beta-adrenergic agonist-stimulated beta-endorphin release and cAMP production in hypothalamic cells in primary cultures; De A et al.; We have previously shown that low concentrations of ethanol rapidly stimulate beta-endorphin (beta-EP) release from hypothalamic neurons in primary cultures and that chronic exposures to these concentrations of ethanol desensitize beta-EP neurons to ethanol challenges . We have also shown that chronic ethanol desensitizes dibutyryl cAMP-, adenosine-, and prostaglandin E1-stimulated beta-EP release and the cAMP content in hypothalamic neurons . In this study, we determined the effects of ethanol (50 mM) on beta-adrenergic agonist (isoproterenol) or alpha-adrenergic agonist (l-phenylephrine)-induced beta-EP release and cellular contents of cAMP to identify whether ethanol causes heterologous desensitization of the adenylate cyclase system in this neuronal cell population . Both isoproterenol and l-phenylephrine increased beta-EP levels in culture media and elevated the cAMP content in cell extracts in a concentration (0.1 and 10 microM)-dependent fashion between 3 to 6 hr . A 50 mM dose of ethanol increased beta-EP and cAMP levels at 3 hr, but it did not elevate beta-EP and cAMP levels after 48 hr of exposure . Acute exposure (3 hr) of these cells to ethanol moderately enhanced the isoproterenol-stimulated and l-phenylephrine-stimulated levels of media beta-EP and intracellular levels of cAMP . However, chronic exposure (48 hr) to ethanol reduced the magnitude of both alpha- and beta-adrenergic receptor agonist-stimulated beta-EP release and cAMP production . These results confirm our previous findings that the ethanol action on beta-EP secretion is mediated by the cAMP system and further suggest that chronic ethanol causes heterologous desensitization of the adenylate cyclase system in the beta-EP neuronal cell population.

J Chromatogr B Biomed Sci Appl, 1999 Jan 8, 721(1), 21 - 9
Identification of the vitamers of vitamin B6 excreted by a yeast mutant growing in a glucose minimal culture medium; Argoudelis CJ; It has been reported that the only vitamers of vitamin B6 excreted by a yeast mutant growing in a fairly complete culture medium were pyridoxine, pyridoxal and pyridoxamine . In this work, evidence is presented that when the same mutant grows in a glucose minimal culture medium it excretes in addition pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate . Differences in the activities of acid phosphatase(s) were found in crude extracts from yeast mutant cells growing in the two culture media.

Biomaterials, 1999 Jan, 20(2), 167 - 73
Osteoblast biocompatibility of mineral trioxide aggregate; Mitchell PJ et al.; This study investigated the biocompatibility of variants of mineral trioxide aggregate (MTA), by culturing human MG63 osteosarcoma cells in the presence of materials, observing cytomorphology and cell growth, and then assaying cytokine expression from the cells . Reference materials were employed . Cell growth was quantified by preparing samples (n = 6) at 2, 4 and 7 days, for viewing by scanning electron microscopy and then scoring the amount of material that was covered by healthy cells . Subsequently, samples of culture media were tested using ELISA assays for expression of Interleukin (IL)-1alpha, IL-6, IL-8, IL-11 and macrophage colony stimulating factor (M-CSF) . These assays were compared with controls where no material was present, and where media and fetal calf serum had not been exposed to cells . Results showed good cell growth on MTA . Expression of IL-6 from cells was only evident in the presence of MTA and Interpore 200 . Interleukin-8 was expressed in high concentrations only in the presence of MTA . There was no evidence of expression of IL-1alpha or IL-11 with any material . Production of M-CSF was high for all materials . It appears that the variants of MTA are biocompatible and suitable for use in clinical trials.

Circulation, 1999 Feb 16, 99(6), 817 - 22
Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes; Matoba S et al.; BACKGROUND--Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level . The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury . METHODS AND RESULTS--Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour . Myocyte injury was determined by the release of creatine kinase (CK) . Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not . The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor) . Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes . In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine . CONCLUSIONS--The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion . NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.

J Neurotrauma, 1999 Jan, 16(1), 27 - 36
Endogenous glutathione protects cerebral endothelial cells from traumatic injury; Gidday JM et al.; Blood-brain barrier breakdown and edema, indicative of cerebrovascular injury, are characteristic pathophysiologic outcomes following head trauma . These injuries result from both primary mechanical damage and from secondary events initiated by the traumatic insult . Free radicals are recognized as mediators of secondary injury in a number of trauma models . In this study, we used a novel in vitro model of traumatic microvascular injury to test the hypothesis that endogenous glutathione protects cerebral endothelial cells from secondary autooxidative injury following mechanical trauma . Porcine brain cerebral endothelial cells were grown in tissue culture wells with Silastic membrane bottoms, and cellular injury was induced by displacing the membrane different distances with user-defined pressure pulses from a customized device . The resultant endothelial cell injury 2 h following stretch was determined by measuring lactate dehydrogenase in the culture media . Significant stretch-dependent increases in endothelial injury were elicited that depended in a nonlinear fashion on the degree of membrane displacement . Depletion of intracellular glutathione with buthionine sulfoximine (1 mM) increased the extent of traumatic endothelial cell injury by 17-56%, particularly at low to moderate levels of traumatic injury (30-40% of total endothelial cell LDH release) . Conversely, traumatic injury was reduced by 22-45% when endothelial cell glutathione levels were augmented threefold (to 140+/-8 nmol/mg protein) by preincubating cells with 2 mM glutathione; the extent of protection was inversely proportional to the extent of the traumatic stretch . Traumatic endothelial cell injury was also significantly and dose-dependently attenuated (up to 40%) by treatment with the xanthine oxidase inhibitor oxypurinol (50 and 100 microM) . These results demonstrate that cerebral endothelial cells are the targets of hydrogen peroxide-mediated injury secondary to trauma-induced superoxide radical formation via the xanthine oxidase pathway . The neutralization of peroxides by the endogenous glutathione redox cycle provides endothelial cells a finite capacity to reduce free radical-mediated traumatic injury; this cycle may be amenable to therapeutic manipulation to mitigate posttraumatic edema and other manifestations of vascular dysfunction.

Am J Trop Med Hyg, 1999 Jan, 60(1), 41 - 50
Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms; Merlen T et al.; The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material . We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species . Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed . Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RPMI 1640 medium for all Leishmania studied . Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media . For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium . Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications.

Adv Dent Res, 1998 Nov, 12(2), 86 - 93
Tetracycline-based MMP inhibitors can prevent fibroblast-mediated collagen gel contraction in vitro; Myers SA et al.; Collagen gels in vitro can be contracted by fibroblasts . The role of matrix metalloproteinases (MMPs) in the contraction of collagen lattices by human neonatal foreskin fibroblasts (HuFFs) was investigated in tissue culture media supplemented by various doses of known gelatinase inhibitors . Fluorescent assays with model gelatinase substrates and media conditioned by fibroblasts apparently confirmed the ability of chemically modified tetracyclines (CMTs) to act as inhibitors of MMP2, and zymography demonstrated that this was the major cell-derived MMP activity . There were no observable effects on the rate of contraction of attached FPCLs containing 6 x 10(4) HuFFs (passages 18-25) with either CMT-5 or CMT-2 at all concentrations tested (0-100 micrograms/mL) . However, at greater than 20 micrograms/mL doxycycline and greater than 5 micrograms/mL CMT-3, FPCL contraction was completely abolished . Quantitative assessment of cell viability by means of the MTT assay in monolayer and qualitatively within the FPCLs with CalceinAM suggested that differences were not due to cytotoxic effects . Seeding FPCLs with lower-passage fibroblasts produced identical trends . These results may implicate the involvement of MMPs in the process of gel contraction, although tetracyclines have effects additional to their ability to inhibit MMPs directly.

Artif Organs, 1999 Jan, 23(1), 114 - 8
The effects of various extracellular matrices on renal cell attachment to polymer surfaces during the development of bioartificial renal tubules; Kanai N et al.; Extracellular matrices (ECM) are utilized for obtaining better cell attachment to polymer surfaces in cell cultures . To establish beneficial bioartificial renal tubules, tubular epithelial cells and ECM were investigated in this study . MDCK cells and KU-2 cells were seeded onto 96 well plates which had been precoated with collagen types I and IV, laminin, and fibronectin . The culture media were removed and replaced with new ones at 15, 30, 60, 90, 120, and 150 min and 24 h after start time to evaluate the incubation time effects . The degrees of cell attachment onto ECM were measured by MTT assay . In the MDCK cell culture, better cell attachment was observed between 60 min and 24 h after incubation start time with the use of laminin at a concentration of 5 microg/ml, 60 min and more after incubation start time with the use of fibronectin at the concentrations of 1 and 4 microg/ml, or 30 min and more after incubation start time with the use of fibronectin at the concentrations of 16 and 32 microg/ml . On the other hand, in the KU-2 cell culture, better cell attachment was observed between 15 and 60 min after the incubation start time or 24 h after the incubation start time with the use of laminin at a concentration of 40 microg/ml . These data suggest that various cells possibly each have a most suitable ECM kind, best concentration, and best incubation time.

Biotherapy, 1998, 11(4), 259 - 65
Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice; Ebina T et al.; The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (10(6) cells) and left flank (2 x 10(5) cells), and were then injected with 5 mg of extracts of A . blazei in the right tumor on days 3, 4 and 5 . Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibition of growth of the left, non-injected tumor . The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments . The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3 . When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 x 10(7) cells/mouse) into the Meth-A tumor, tumor growth was inhibited . The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity . Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue . Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3 . These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

Rev Argent Microbiol, 1998 Oct-Dec, 30(4), 195 - 9
{Growth in species of the genus Ascobolus (Pezizales-Ascomycetes)}; Dokmetzian D et al.; The kinetics of growth of eight heterothallic species of the genus Ascobolus was studied in liquid culture media . The results obtained showed variation among the species in the duration of the different phases of the growth cycle . Three groups can be recognized considering the extension of the exponential phase of growth . The stationary phase, which differs in its length, is frequently very short, entering quickly in the phase of death, accompanied by autolysis of the mycelium.

Glycobiology, 1999 Feb, 9(2), 125 - 31
Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells; Gawlitzek M et al.; Elevated ammonium concentrations in the medium of cultivated cells have been shown to increase the intracellular levels of uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994) . These sugar nucleotides are substrates for glycosyltransferases in the glycosylation pathway . In our experiments, recombinant Chinese hamster ovary cells producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated under controlled cell culture conditions in the presence of different ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added exogenously to the cell culture media to determine if ammonium was incorporated into UDP-GlcNAc and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently incorporated into GP1-IgG as N-linked glycans . The intracellular pools of UDP-activated hexosamines (UDP-GNAc) were followed during the time course of the experiment . To assess the extent of15NH4+incorporation into the glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by protein A chromatography . Enzymatically released N-glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . N-Glycans synthesized in the presence of15NH4Cl revealed an N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da . These results indicate that 60-70% of the total nitrogen containing monosaccharides had incorporated15N . Presumably,15NH4+was incorporated into GlcNAc and N-acetylneuraminic acid as proposed earlier (Ryll et al., 1994) . This might be a universal and previously not described reaction in mammalian cells when exposed to nonphysiological but in cell culture commonly found concentrations of ammonium . The data presented here are of significance for glycoprotein production in mammalian cell culture, since it has been shown previously that elevated levels of UDP-activated hexosamines affect N-glycan characteristics such as branching and degree of amino sugar incorporation . In addition, our results demonstrate that isotope labeling in combination with MALDI-TOF-MS can be used as an alternate tool to radioactive labeling of sugar substrates in metabolic studies.

Cell Tissue Res, 1999 Feb, 295(2), 297 - 305
Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages; Cervar M et al.; In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion . Trophoblast was isolated from 17 term placentas (-IP) . One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody . This increased the proportion of trophoblast (+IP >97%; -IP approximately 70%) as identified by rigorous immunocytochemistry . Most (approximately 70%) non-trophoblast cells in -IP were macrophages . The cells were cultured for 5 days with a daily medium change . In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media . The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-beta (hCG-beta) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media . The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-beta: 18-fold) in +IP than in -IP, with the exception of endothelin-1,2 (no change), angiotensin II (-70%) and 6-keto-prostaglandin-F1alpha (-40%) . {3H}leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in -IP . Addition of conditioned media reverted these changes . The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion . We conclude that macrophages are important regulators of trophoblast activity.

J Mol Med, 1999 Jan, 77(1), 90 - 2
Glucose sensitivity of porcine and human islets in vitro; Brandhorst H et al.; Preliminary experiments about the suitability of different commonly used culture media in our laboratory indicated, that prolonged exposure to high glucose concentrations during low temperature culture (LTC) impairs the viability of long term cultured human islets . As a consequence of the heterogeneity of tested media the present study was aimed to evaluate the influence of different glucose concentrations on survival, viability and in-vitro function of cultured human islets in order to optimize islet survival until transplantation and to compare species dependent differences in glucose sensitivity . Quantified aliquots of freshly isolated (digestion-filtration, ficoll gradient purification) islets from consecutively processed human (n=6) and porcine (n=11) pancreata were subjected to different glucose concentrations (human islets: 500, 750, 1000 and 2000 mg/l; porcine islets: 1000 and 2000 mg/l) in CMRL (22 degrees C) for 8-10 days . After LTC survival, viability and glucose-stimulated insulin release of incubated tissue was assessed . A reduction of glucose concentration promotes survival and viability of human islets but impairs in vitro function at the same time, presumably due to a reduced glucose oxidation as expressed by the significantly reduced stimulation index . In contrast to these findings in the human, elevated glucose concentration in porcine islet culture increases survival but reduces the glucose-stimulated insulin release and the viability of cultured islets . The contradiction of the results in regard to islet survival related to islet viability are still unclear in the pig and needs further evaluation.

Matrix Biol, 1998 Dec, 17(8-9), 657 - 65
A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjögren's syndrome contain increased latent and active gelatinase-B levels; Hanemaaijer R et al.; Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay . Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys/IleIleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly/IleIleGlyGly) . The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase . The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody . Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed . We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjogren's syndrome . Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 +/- 4.9 vs 12.2 +/- 2.5 x 10(4) cpm/ml (p > 0.05, and 44.0 (4.0 vs 36.1 +/- 1.9 x 10(4) cpm/ml (p > 0.05), for active and latent gelatinase, respectively . However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 +/- 2.5 U/mg, controls 1.0 +/- 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 +/- 9.8 U/mg, controls 16.5 +/- 2.6 U/mg (p = 0.01) . This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.

Eur J Clin Microbiol Infect Dis, 1998 Nov, 17(11), 767 - 72
Comparison of the ligase chain reaction with solid and liquid culture media for routine detection of Mycobacterium tuberculosis in nonrespiratory specimens; Palacios JJ et al.; The aim of this study was to compare the results of a commercial assay based on the ligase chain reaction {(LCR) LCx Probe System MTB; Abbott, USA} with those of culture in liquid medium (Septi-Chek AFB; Becton-Dickinson, USA) and culture on the egg-based Lowenstein-Jensen solid medium for the direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens . The results were analyzed according to the standard definition of a true-positive result . Two hundred thirty-five nonrespiratory samples routinely submitted to rule out tuberculosis were analyzed . All samples were smear-negative . Mycobacterial growth in either culture medium was detected in 18 (7.6%) specimens: Mycobacterium tuberculosis was recovered from seven (38.9%) specimens cultured on Lowenstein-Jensen medium and from 18 (100%) specimens cultured in Septi-Chek AFB . The LCR protocol was positive in 22 specimens . None of the LCR-negative controls showed positive results . All samples positive by culture on Lowenstein-Jensen medium were positive by culture in liquid medium and by the LCR assay . However, Mycobacterium tuberculosis was detected by culture in liquid medium in two specimens that were negative by the LCR assay, whereas six specimens negative by culture in liquid medium were positive by the LCR protocol; three of these were identified as true-positive results of the LCR assay . The sensitivity, specificity, and positive and negative predictive values were 33.3%, 100%, 100%, and 93.8%, respectively, for Lowenstein-Jensen medium; 85.7%, 100%, 100%, and 98.6% for the liquid medium; and 90.4%, 98.5%, 86.3%, and 99% for the LCR assay . These findings indicate that the LCR assay may be a valid method of high diagnostic yield for direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens.

Can J Physiol Pharmacol, 1998 Jun, 76(6), 621 - 9
Long-term supplementation of culture medium with essential fatty acids alters alpha-linolenic acid uptake in Caco-2 clone TC7; Tranchant T et al.; We investigated the influence of four different culture media: 20% fetal bovine serum (FBS), 5% FBS, 5% FBS supplemented with 10 mg x L(-1) linoleic acid (18:2(n-6)) or alpha-linolenic acid (18:3(n-3)) on alpha-linolenic acid apical uptake in clone TC7 of human intestinal Caco-2 cell line . Neither cellular viability nor cell monolayer integrity and permeability were altered by the four culture conditions . Our results show that the different culture media led to changes in alpha-linolenic acid maximal rate of uptake (Vmax) but did not alter the apparent transport constant (Km) . Reducing FBS concentration from 20% to 5% increased significantly the rate of alpha-linolenic acid uptake, which was further increased by supplementation of the medium with 18:2(n-6) or 18:3(n-3) . Supplementation with essential fatty acids led to a marked enrichment of brush-border membrane phospholipids in polyunsaturated fatty acids of the corresponding series and decreased significantly the levels of monounsaturated fatty acids . Saturated fatty acids, unsaturation index, and cholesterol/fatty acid ratios were unchanged . No clear relation could be established between the changes in membrane lipid composition and the alterations of alpha-linolenic acid uptake . These results indicate a weak influence of membrane lipid composition in the modulation of the uptake . Therefore, the increase of uptake following long-term supplementation of TC7 cells with essential fatty acids could be attributed to an increase of the expression of membrane protein(s) involved in the apical uptake of long-chain fatty acids . This remains to be established.

Thyroid, 1998 Dec, 8(12), 1157 - 63
Thyroid hormone inhibits aromatase activity in porcine thecal cells cultured alone and in coculture with granulosa cells; Gregoraszczuk EL et al.; We cultured porcine thecal and granulosa cells alone or in coculture to define whether thyroid hormone affects aromatase activity in porcine ovarian cells . Dispersed cells were cultured with 10(-9) M triiodothyronine (T3) for 24 hours . Testosterone (final concentration 10(-7) M) was added as aromatase substrate for granulosa cells (Gc) cultured alone . Thecal (Th) androgens serve as a substrate for estradiol secretion by Th cells cultured alone and in coculture with Gc . At the end of the preincubation time, the culture media was removed and replaced with fresh media containing 100 ng follicle stimulating hormone (FSH) or 10(-3) M 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) . After overnight incubation, the culture media was analyzed for estradiol production by radioimmunoassay (RIA) . T3 inhibited basal, FSH-stimulated, and 8brcAMP-stimulated estradiol production in all culture conditions . T3 inhibited cAMP analogue 8-Br-cAMP and FSH-induced aromatase activity to a similar extent, thus suggesting that the inhibitory effect of T3 is downstream of cAMP formation . In the second part of the experiment a rabbit polyclonal antibody against human placental cytochrome P-450arom was used to confirm the effect of T3 on aromatase protein in Th and Gc . Pretreatment of Th and Gc with T3 markedly decreased immunostaining for aromatase in both types of cells, suggesting a direct effect of T3 on this enzyme.

J Parasitol, 1998 Dec, 84(6), 1267 - 71
Improved culture media for piscine hemoflagellates, Cryptobia and Trypanosoma (Kinetoplastida); Ardelli BF et al.; Increasing the Hepes buffer in minimum essential medium from 25 mM to 100 mM yielded a significantly larger number of Cryptobia salmositica . Cryptobia salmositica (pathogenic and nonpathogenic strains), Cryptobia bullocki, and Trypanosoma danilewskyi did not multiply either in heat-inactivated trout plasma (< or =25%) or in less than 10% fresh trout plasma . Both strains of C . salmositica multiplied better in 10% fresh trout plasma than in 25% heat-inactivated fetal bovine serum . In contrast, C . bullocki and T . danilewskyi multiplied better in 25% fetal bovine serum; 10% fetal bovine serum did not significantly reduce multiplication of C . bullocki . The nonpathogenic vaccine strain of C . salmositica cultured in 10% fresh trout plasma still protected rainbow trout from high parasitemia when challenged with the pathogen.

Jpn J Pharmacol, 1998 Dec, 78(4), 429 - 34
Effect of ambroxol on oxygen radical production and generation by bronchoalveolar lavage cells in young and aged guinea pigs; Teramoto S et al.; We examined the effect of ambroxol and age on oxygen radical production and generation with stimulation of phorbol-myristate acetate (PMA) by bronchoalveolar lavage (BAL) cells . Lung free cells including pulmonary alveolar macrophages were harvested from young (4-month-old) and aged (28-month-old) male guinea pigs using BAL . The oxygen radicals produced by BAL cells with or without stimulation of PMA were measured by the lucigenin-dependent chemiluminescence method using a photon counter . Oxygen radical production and generation by BAL cells were not different between young and aged guinea pigs . However, the oxygen radical generation after stimulation with PMA was greater than the oxygen radical spontaneous production both in young and aged animals . Ambroxol solution given into culture media containing BAL cells inhibited oxygen radical production and generation by BAL cells harvested from both young and aged guinea pigs in a concentration-dependent manner . Approximately 16-20 microM of ambroxol inhibited 50% of the production of oxygen radicals in vitro by BAL cells in young and aged guinea pigs, whereas a slightly greater amount of ambroxol was necessary to inhibit 50% of the PMA-induced oxygen radical generation in vitro by BAL cells in guinea pigs . These results indicate that ambroxol inhibits oxygen radicals produced by BAL cells from young and aged guinea pigs, and they suggest that ambroxol may be a possible therapeutic modality for ameliorating oxidant associated pulmonary disorders in young and aged patients.

Arthritis Rheum, 1999 Jan, 42(1), 137 - 47
Retinoic acid-induced type II collagen degradation does not correlate with matrix metalloproteinase activity in cartilage explant cultures; Price JS et al.; OBJECTIVE: To determine the role of matrix metalloproteinases (MMPs) in retinoic acid (RetA)-induced degradation of type II collagen in cartilage . METHODS: Bovine nasal cartilage explants were cultured with 1 microM RetA or in 3 nM interleukin-1alpha (IL-1alpha) . Release of proteoglycan and type II collagen into the medium was measured by colorimetric assay and immunoassay, respectively . MMP activity in the medium was determined using a quenched fluorescent substrate assay, while specific collagenases were identified by Western immunoblotting . In some cases the effects of low molecular mass synthetic MMP inhibitors and serum on collagen degradation were studied . RESULTS: RetA promoted maximal breakdown of type II collagen after 4 or 5 weeks in culture, compared with 3 weeks in culture with IL-1alpha . In IL-1alpha-stimulated cultures, collagen degradation was coincident with a large increase in MMP activity in the culture medium, whereas in RetA-stimulated cultures, there was only a small increase . In Western immunoblots of culture media containing RetA, prointerstitial collagenase and active collagenase 3 were sometimes detected, but not in all experiments . In IL-1alpha cultures, active interstitial collagenase was always detected, and active collagenase 3 was detectable in some experiments . Neutrophil collagenase was not detected in any cultures . IL-1alpha-stimulated collagen degradation was effectively inhibited by a potent, broad-spectrum inhibitor of MMPs, whereas it was poorly inhibited by a weak MMP inhibitor . The same 2 compounds were both only weak inhibitors of RetA-induced collagen degradation . When fetal calf serum was included in cartilage cultures, MMP activity in the culture medium was reduced to low levels . This resulted in a marked inhibition of IL-1alpha-induced type II collagen degradation, whereas there was no inhibition of RetA-induced collagen degradation . CONCLUSION: Unlike IL-1alpha, RetA induces degradation of type II collagen in cartilage explants by a mechanism that is mainly independent of those MMPs that can be detected in the culture medium.

Mycoses, 1998 Dec, 41(11-12), 529 - 33
Euphorbia hirta leaves and Musa sapientum fruits in culture media for fungi; Emele FE et al.; Two plant products, Euphorbia hirta leaves and fruits of Musa sapientum, were evaluated as principal ingredients for selective cultivation of fungi . Sapientum glucose agar supported the growth of both dermatophytic, yeast-like, and saprophytic fungi; growth on this medium compared favourably with growth on Sabouraud glucose agar, a standard mycological medium . Sporulation and pigment formation were stronger on sapientum glucose agar than on Sabouraud glucose agar, although fungal growth on the latter was more luxuriant . Addition of Euphorbia extract to mycological media remarkably enhanced fungal growth on the media, and concomitantly suppressed bacterial growth to a similar extent as did antibiotics . The results of this study suggest that Euphorbia sapientum glucose agar can safely be recommended as a cheap and efficient medium for routine isolation of fungi in both clinical and general mycological studies.

Biochem Biophys Res Commun, 1999 Jan 19, 254(2), 462 - 5
Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes; Seko Y et al.; Evidence has accumulated that vascular endothelial growth factor (VEGF) is expressed in the heart, and its expression is markedly increased in response to hypoxia . Recently, it was shown that pulsatile myocardial stretch in vivo markedly enhanced VEGF mRNA level in the heart . To investigate whether pulsatile mechanical stretch really stimulates VEGF expression by cardiac myocytes, using an in vitro preparation, we examined the secretion of VEGF into the culture media from cardiac myocytes subjected to pulsatile stretch . We found that pulsatile mechanical stretch induced rapid secretion of VEGF by cultured rat cardiac myocytes and mRNA expression of VEGF and VEGF receptors in the cardiac myocytes . We also found that the stretch-induced secretion of VEGF was at least in part mediated by TGF-beta . These data provide the direct evidence that mechanical overload itself can induce VEGF secretion by cardiac myocytes, which may play a role in ameliorating the relative myocardial hypoxia .

Dev Biol, 1999 Feb 1, 206(1), 88 - 99
Human mammary luminal epithelial cells contain progenitors to myoepithelial cells; Pechoux C et al.; The origin of the epithelial and myoepithelial cells in the human breast has not been delineated . In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other . We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture . The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity . The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis . Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and alpha-smooth muscle actin . We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor . The two different culture media supported each lineage for at least five passages without signs of interconversion . We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other . Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells . We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around .

Circulation, 1999 Jan 26, 99(3), 420 - 6
Interleukin-8 mediates downregulation of tissue inhibitor of metalloproteinase-1 expression in cholesterol-loaded human macrophages: relevance to stability of atherosclerotic plaque; Moreau M et al.; BACKGROUND: The accumulation of macrophage-derived foam cells in atherosclerotic lesions correlates with increased local release of matrix-degrading metalloproteinases (MMPs) and a thin fibrous cap . The activity of these enzymes is controlled by specific tissue inhibitors of metalloproteinases (TIMPs) . METHODS AND RESULTS: Because oxidized low-density lipoprotein (OxLDL) modulates gene expression, we investigated the effect of these particles on the levels of MMP-1, MMP-3, MMP-9, TIMP-1, and TIMP-2 in the culture media of human monocyte-derived macrophages . OxLDL but not native LDL or high-density lipoprotein reduced the level of TIMP-1 in a dose-dependent manner with maximal effect (60% of control) at approximately 100 microg protein/mL . In addition, Northern blotting revealed marked reduction in the abundance of TIMP-1 mRNA in OxLDL-treated cells . Evaluation of the effect of oxysterol components of OxLDL on TIMP-1 production revealed that 25-hydroxycholesterol (1 microg/mL) was the most potent inhibitor ( approximately 30% of control) . Such inhibition was partially mediated by interleukin (IL)-8 . Indeed, IL-8 (2.5 ng/mL) induced maximal inhibition of TIMP-1 accumulation (30% of control) in 4 of 6 cell preparations . In addition, the inhibitory effect of OxLDL-treated cells in the presence of an anti-IL-8 neutralizing antibody was partially reversed . CONCLUSIONS: Immunohistochemical analyses of human atherosclerotic plaques revealed the expression of TIMP-1 in some but not all macrophage-rich and IL-8-rich areas . Therefore, IL-8 may play a potential atherogenic role by inhibiting local TIMP-1 expression, thereby leading to an imbalance between MMPs and TIMPs at focal sites in the atherosclerotic plaque.

Ann N Y Acad Sci, 1998 Sep 11, 858, 147 - 62
Response of a liver tissue slab to a hyperosmotic sucrose boundary condition: microscale cellular and vascular level effects; Bhowmick S et al.; Transport of a non-permeating CPA in liver tissue was studied by experimental and theoretical techniques . The system consisted of a 20 mm x 15 mm x 500 microns (thick) slab of liver tissue which was exposed to culture media and hyperosmotic sucrose (0.3 or 0.6 M) at the boundary . The volumetric changes of cell and vascular spaces within the tissue slab at 125 microns from one of the symmetric boundaries was studied by slam freezing followed by freeze substitution microscopy . The experimental data was then theoretically investigated using two models; one based on an effective diffusion coefficient for sucrose, and another which incorporated the convective flux of water out of the cells (and the tissue) while sucrose diffuses in . We estimate the effective diffusion of sucrose as 16-33% of the actual diffusivity of sucrose in bulk water . The role of convection of water out of the tissue is against the flow of sucrose and appears to be important in reducing the effective diffusivity of the sucrose . The role of vascular compliance, porosity and tortuosity are also discussed with respect to our results.

Infect Immun, 1999 Feb, 67(2), 853 - 61
Expression and distribution of leptospiral outer membrane components during renal infection of hamsters; Barnett JK et al.; The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41 . The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection . Hamsters were challenged with host-derived Leptospira kirschneri to generate sera which contained antibodies to antigens expressed in vivo . Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36 . Although LipL36 is a prominent outer membrane antigen of cultivated L . kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo . In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection . Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS . When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes . These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.

Cornea, 1999 Jan, 18(1), 92 - 7
Bacterial lipopolysaccharides in sterile corneal organ-culture media; Sobottka Ventura AC et al.; PURPOSE: Lipopolysaccharides (LPS) are known to stimulate various inflammatory reactions by interaction with cytokines and macrophages . As contamination of sterile organ culture media with nonviable bacterial substances may influence donor tissue prognosis, we investigated a series of culture media drawn from organ culture for the presence of LPS . METHODS: One hundred eighty-two samples of sterile organ-culture media were tested for LPS using the Limulus-amoebocyte-lysate assay (LALA) . We then investigated the time course of LPS levels during organ culture, the influence of medium changes, the graft deswelling procedure and transportation as well as repeated freezing on the detection of lipopolysaccharides with the LALA . RESULTS: LPS above background threshold was found in 21.4% of the organ-culture media . The time course of LPS during organ culture and through the deswelling procedure was quite stable . Medium changes may wash out LPS, thus the highest LPS values were normally seen in the examination medium, which has the first contact with the corneal tissue . Repeated freezing did not influence the detectability of LPS with the LALA . CONCLUSION: LPS detected in sterile corneal organ cultures is probably derived from nonreplicating bacterial postmortem donor tissue contamination . It is a rather heat and cold stable product that may be washed out from the donor tissue by medium changes . As LPS may directly influence graft viability or trigger inflammatory host responses, further investigations of the clinical course of these grafts are required.

J Biochem Mol Toxicol, 1999, 13(1), 11 - 5
The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells; Forman S et al.; Testing of the effects of xenobiotics in cultured cells often requires the use of organic solvents to effect suspension of the test agents in cell culture media . However, the toxic effects of the solvents themselves may introduce artifacts, which obscure interpretation of the experimental results . In this article, the toxicity of different solvents commonly used for solvation of a variety of xenobiotic agents was studied . We show that ethanol, acetone, isooctane, methanol, and hexane were considerably less toxic than the more commonly used solvent, DMSO, when ATP content and growth rates of HeLa cells exposed to these solvents was measured.

J Biochem Mol Toxicol, 1999, 13(2), 83 - 91
Linoleic acid amplifies polychlorinated biphenyl-mediated dysfunction of endothelial cells; Hennig B et al.; Selected dietary lipids may increase the atherogenicity of environmental chemicals, such as polychlorinated biphenyls (PCBs), by cross-amplifying mechanisms leading to dysfunction of the vascular endothelium . To investigate this hypothesis, cultured endothelial cells were treated with 90 microM linoleic acid (18:2n-6), followed by either one of two PCBs, 3,3',4,4'-tetrachlorobiphenyl (PCB 77) or 2,2'4,4',5,5'-hexachlorobiphenyl (PCB 153) . These PCBs were selected for their varying binding activities with the aryl hydrocarbon (Ah) receptor and differences in their induction of cytochrome P450 . PCB 77 disrupted endothelial barrier function by allowing an increase in albumin transfer across endothelial monolayers . Prior cellular enrichment with 18:2 before PCB treatment further diminished endothelial barrier function, as compared to cells treated only with the PCB . This phenomenon appears to be mediated by increased oxidative stress, which is supported by enhanced 2,7-dichlorofluorescein fluorescence, activation data of the oxidative stress-sensitive nuclear transcription factor-kappaB (NF-kappaB), as well as an observed decrease in vitamin E content in the culture media . Similar to the endothelial permeability data, pre-enrichment of cells with 18:2 further increased the PCB-mediated induction of cytochrome P450 1A . In contrast to PCB 77, PCB 153 (or 18:2 plus PCB 153) had little or no effect on endothelial barrier function . Our results suggest that certain unsaturated fatty acids can potentiate PCB-mediated endothelial cell dysfunction and that oxidative stress and activation of the cytochrome P450 1A subfamily may be, in part, responsible for these metabolic events . These findings have implications for understanding the involvement of certain environmental contaminants in diseases that involve dysfunction of the vascular endothelium.

J Clin Microbiol, 1999 Feb, 37(2), 283 - 9
Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method; Buonagurio DA et al.; A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine . The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media . Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result . Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation . The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively . In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively . Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination . In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks . Shedding of type 3 virus ceased in approximately 70% of vaccinees within a week after immunization . In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Am J Physiol, 1999 Jan, 276(1 Pt 2), F129 - 36
Obstruction stimulates COX-2 expression in bladder smooth muscle cells via increased mechanical stretch; Park JM et al.; Studies were performed to investigate the regulatory mechanism of bladder cyclooxygenase-2 (COX-2) expression after outlet obstruction . In situ hybridization of murine bladder tissues using COX-2-specific riboprobes demonstrated that COX-2 expression was induced predominantly in the bladder smooth muscle cells after outlet obstruction . To study the effect of increased mechanical stretch on COX isoform expression, cultured rat bladder smooth muscle cells were grown on silicone elastomer-bottomed plates coated with collagen type I and were subjected to continuous cycles of stretch/relaxation for variable duration . COX-1 mRNA levels did not change with stretch . COX-2 expression increased in a time-dependent manner after stretch, with maximal mRNA and protein levels occurring after 4 h . PGE2 levels increased more than 40-fold in the culture media after stretch, consistent with increased COX activity, and this was reduced to near completion in the presence of a COX-2 inhibitor, NS-398 . Exposure to stretch over a 48-h period induced a 4.7 +/- 0.6-fold increase in tritiated thymidine incorporation rate . This increase in DNA synthesis was markedly suppressed when the cells were stretched in the presence of NS-398 . We conclude that in bladder obstruction COX-2 activation occurs predominantly in the smooth muscle cells in response to mechanical stretch . Our findings also suggest that stretch-activated COX-2 expression may participate in bladder smooth muscle cell proliferation and thereby play a role in pathological bladder wall thickening after obstruction.

Hum Reprod, 1998 Dec, 13(12), 3441 - 8
Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH; Edwards LJ et al.; The inclusion of specific amino acids in conventional culture media has been shown to enhance mammalian embryo development in vitro . Amino acids have been shown to confer their benefits to the preimplantation embryo in a number of different ways . However, their ability to buffer intracellular pH (pHi) has not been investigated . Thus, the aim of this study was to determine if amino acids regulate pHi in the mouse preimplantation embryo . pHi was determined using carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy . Incubation with 5,5-dimethyl-2,4-oxazol-idinedione (DMO), a non-metabolizable weak acid, resulted in a significant intracellular acidification in the zygote, 2-, 4- and 8-16-cell embryo . However, in the presence of groups of amino acids, the degree of acidification due to DMO was markedly reduced in the mouse embryo up to the 4-cell stage . Specifically, non-essential amino acids and glutamine had the greatest capacity to buffer pHi in the early embryo . The ability of amino acids to buffer pHi was not apparent from the 8-16-cell stage onwards . In contrast to the precompacted embryo, the morula did not undergo a significant decrease in pHi until exposed to DMO concentrations > or = 10 mM in the absence of amino acids . This may be due to the generation of a permeability seal during compaction, thus enabling the morula to regulate its own pHi . This regulatory ability could either be reversed by causing the morula to decompact, or created by inducing premature compaction in the 8-16-cell embryo . Data presented in this study indicate that amino acids act as buffers of pHi in the early embryo and play a key role in regulating cell physiology . Further evidence for this was provided by the result that only those embryos cultured in 30 mM DMO in the presence of non-essential amino acids and 1 mM glutamine did not block at the 2-cell stage, but grew on to develop into expanded blastocysts.

Endocr J, 1998 Aug, 45(4), 467 - 73
Effect of site directed mutagenesis in the CMGCC region of the alpha-subunit on immunoreactive human thyrotropin; Miyai K et al.; The cDNA of the common alpha-subunit of human glycoprotein hormone was mutated by site directed mutagenesis in the CMGCC region composed of cysteine-methionine-glycine-cysteine-cysteine (position 28-32) . The cDNA of wild-type human thyrotropin (hTSH) beta-subunit and that of wild-type or mutant common alpha-subunits were co-transfected into COS-I cells . The concentration of hTSH determined by two immunoradiometric assay systems was detectable in culture media of COS-I cells transfected with wild-type (CMGCC) and a mutant (CRGCC) alpha-subunits but not four other mutants (YMGCC) (CMRCC) (CMACC) (CMDCC) . The present data with the other studies on wild-type or mutant glycoprotein hormones support our hypothesis that an amino acid motif of "C-X-G-X-C" in the common alpha-(CMGCC in human) and beta-(CAGYC in human) subunits play an important role in biosynthesis of glycoprotein hormones in all species.

Endocr J, 1998 Aug, 45(4), 441 - 50
Co-expression of transforming growth factor beta and interferon tau during peri-implantation period in the ewe; Imakawa K et al.; The transforming growth factor beta (TGFbeta) family is known to control cell migration, growth, differentiation, function and regulation of extracellular matrix, all of which are required for the process of implantation . Expression of TGFbeta by the conceptus and endometrium was studied during the period of implantation in the ewe . A total of thirty-four ewes were hysterectomized on day 12, 14, 16, 18 or 20 of pregnancy (day 0 = day of estrus) . Conceptus (200 mg wet weight) and endometrial (300 mg wet weight) tissues were cultured in vitro in 7 and 10 ml Eagle's minimal essential medium, respectively . The culture media were subjected to a bioassay to determine concentrations of TGFbeta . Conceptus culture media (CCM) were also analyzed for contents of ovine interferon-tau (oIFNr), low molecular weight acidic protein, produced by the trophectoderm between days 8 and 21 of pregnancy . Whole uteri including conceptus(es) and conceptuses (day 16) only were fixed and subjected to immunohistochemical and in situ hybridization studies . Levels of oIFNr produced by conceptuses were the highest on day 16 at 4.4 microg/ml . Concentrations of TGFbeta in day 12, 14, 16, 18 and 20 CCM were 38+/-19, 102+/-56, 862+/-152, 728+/-191 and 336+/-106 pg/ml, respectively, and approximately 90% of TGFbeta activity in CCM was due to TGFbeta1 whereas less than 10% was due to TGFbeta3 based on neutralization with TGFbeta subtype-specific antibodies . Immunohistochemical studies revealed that day 16 conceptuses displayed major staining for TGFbeta1, no beta2 staining and minor staining for beta3 . In situ hybridization studies also revealed that day 16 trophectoderm possessed most TGFbeta1 mRNA while day 14 trophectoderm and day 20 chorion/amnion displayed weaker staining for TGFbeta1 mRNA . TGFbeta in day 12, 14, 16, 18 and day 20 endometrial culture media was 156+/-37, 129+/-33, 49+/-22, 62+/-23 and 179+/-40 pg/ml, respectively, and approximately 65% and 35% of the activities were due to TGFbeta1 and beta2, respectively . These results indicate that TGFbeta production by the conceptus coincides with the time when oIFNtau production starts to decline . These observations support the postulate that TGFbeta may play an important role in implantation in the ovine species.

Biol Pharm Bull, 1998 Dec, 21(12), 1267 - 70
Soluble factors from rat basophilic leukemia (RBL-2H3) cells stimulated cooperatively the neurite outgrowth of PC12 cells; Suzuki M et al.; Culture media from rat basophilic leukemia cells (RBL-2H3) induced the neurite outgrowth of rat pheochromocytoma PC12 cells, a model system for neuronal differentiation . The extension of the neurite outgrowth was dependent on the culture time of RBL-2H3 cells in the DMEM medium . The DMEM medium conditioned by RBL-2H3 cells for 48 h induced neurite outgrowth of PC12 cells significantly . The neurite extension was much higher than that by medium containing 1 ng/ml nerve growth factor (NGF) but was rather lower than that by medium containing 10 or 50 ng/ml NGF . The neurite extension by 50 ng/ml NGF was completely suppressed by excess anti-NGF antibody (1-1.5 microg/ml), while the extension by culture medium conditioned by RBL-2H3 cells for 48 h was not completely suppressed in the presence of the same amount of anti-NGF antibody . The neurite extension by the culture medium of RBL-2H3 cells was also suppressed by anti-interleukin (IL)-6 antibody (1 microg/ml), although IL-6 itself (20 units) could scarcely induce the neurite outgrowth of PC12 cells . This suggests that IL-6 in the culture medium of RBL-2H3 cells could be effective in inducing the neurite extension in cooperation with NGF . In the presence of an excess of both anti-NGF and anti-IL-6 antibodies, the culture medium of RBL-2H3 cells induced the neurite extension of PC12 cells . This suggests that the action of the various factors from RBL-2H3 cells may be synergistic as far as the neurite outgrowth of PC12 cells is concerned.

Brain Res, 1999 Jan 9, 815(2), 389 - 99
Cocaine adversely affects development of cortical embryonic neurons in vitro: immunocytochemical study of calcium-binding proteins; Glezer II et al.; Neurons of cerebral cortex from 15-16 day old embryos of white rats (Sprague-Dawley) were cultured in MEM enriched with 5% horse serum . On the 7th day after plating the cultures were divided into three experimental and one control groups (6-8 Petri dishes in each group) . In group 1, cultures were grown without additives . In group 2, cocaine chloride was added at concentrations 0.3, 0.6 and 1 mg/ml of culture . In group 3, a monoclonal antibody against calcium-binding proteins, parvalbumin (APV) or calbindin (ACB) was added at a concentration 25 microl/ml . In group 4, a combination of cocaine +APV was added at a concentration 1 mg+25 microl/ml of culture media . On the 10th day cultures were immunostained using APV and ACB antibodies . In developing GABAergic neurons of group 2 cocaine produced cytotoxic effects that were expressed in drastic decrease in number of neurons and in degeneration of their processes . The lower concentrations of cocaine caused milder cytotoxity and their effects were reversible . The highest concentration of cocaine caused irreversible degeneration of neurons . Similar cytotoxity was caused by APV or ACB in group 3 . The most severe cytotoxic effects were seen in group 4, where a mixture of cocaine and APV was used . Overall, it can be concluded that cocaine in higher concentrations directly affects development of GABAergic neurons in vitro .

Clin Endocrinol (Oxf), 1998 Oct, 49(4), 465 - 73
The IGF-I/IGFBP system in congenital partial lipodystrophy; Janssen JA et al.; BACKGROUND AND OBJECTIVES: Insulin and IGF-I interact at many levels . Little is known about the insulin-like growth factor-I/insulin-like growth factor binding proteins (IGF-I/IGFBP) system in congenital partial lipodystrophy, a syndrome characterized by insulin resistance, hyperinsulinaemia and absence of truncal and limb fat . Some cases have acromegaloid features with thick skin and large hands and feet in association with normal levels of circulating growth hormone . METHODS: In four females known with congenital partial lipodystrophy, hyperinsulinaemia with acromegaloid features, the number and affinity of the IGF-I receptors on peripheral blood mononuclear cells (PBMCs), and the concentration of circulating insulin, total and free IGF-I, IGFBP-1 and IGFBP-3 levels were measured in the fasting and the fed state . Cultures of PBMCs of the patients with lipodystrophy were also used to study the effect of IGF-I stimulation on thymidine uptake in vitro . MEASUREMENTS: In the subjects with lipodystrophy the affinity and the number of the IGF-I receptors on peripheral mononuclear cells (PBMCs) and erythrocytes did not differ significantly from controls in the fasting state . Insulin levels were significantly higher in subjects with lipodystrophy both in the fasting as well in the fed state . Total IGF-I, free IGF-I and IGFBP-3 levels did not differ but serum IGFBP-1 levels were lower in lipodystrophy subjects than in healthy controls . The free IGF-I/IGFBP-1 ratio was increased in lipodystrophy subjects both in the fasting and the fed states . The effects of IGF-I stimulation on thymidine uptake by PBMCs of lipodystrophy subjects in the absence of IGFBP-1 were not different from healthy controls cultures in vitro . When a combination of IGFBP-1 (in a concentration comparable to the fasting serum IGFBP-1 levels in lipodystrophy patients found in our study) and IGF-I was added to PBMC cultures from lipodystrophy patients no decrease in thymidine uptake by PBMCs was found . CONCLUSIONS: In the four subjects with lipodystrophy hyperinsulinaemia, lowered free IGF-I and IGFBP-1 levels, but increased free IGF-I/IGBP-1 ratios were observed . Low IGFBP-1 concentrations in culture media did not reduce the stimulating IGF-I effect on thymidine uptake by PBMCs from lipodystrophy patients . Our data suggest that the observed increased IGF-I/IGFBP-1 ratio in lipodystrophy patients contributes to an unopposed biological effect of IGF-I on IGF-I receptors, thereby inducing the development of acromegaloid features, acanthosis nigricans and polycystic ovaries in some patients with congenital partial lipodystrophy.

Biochem Biophys Res Commun, 1998 Dec 9, 253(1), 170 - 5
Molecular cloning and functional expression of a fifth-type alpha 2,3-sialyltransferase (mST3Gal V: GM3 synthase); Kono M et al.; The cDNA encoding a new type of alpha 2,3-sialyltransferase (mST3Gal V) was cloned from mouse brain cDNA library by PCR-based cloning approach using a pair of degenerate primers deduced from the nucleotide sequence information of mouse ST3Gal III and IV . The predicted amino acid sequence of mST3Gal V showed 27.3% and 26.4% identity to mST3Gal III and IV, respectively . The recombinant soluble mST3Gal V fused with protein-A, which expressed in the culture media of COS-7 cells, showed activity toward lactosylceramide (LacCer), and synthesized GM3 . The apparent Km value for LacCer was 9.3 microM . mST3Gal V did not exhibit any activity toward other substrates we tested in this study, including glycolipids, glycoproteins and disaccharides . The mST3Gal V cDNA transfected F28-7 cells, which express large amount of lactosylceramide and very small amount of GM3 at native stage, expressed a large amount of GM3 . The ST3Gal V gene was strongly expressed in mouse brain and liver, which contained a large amount of ganglioside . The gene expression seemed to be coincident with ganglioside expression in mouse . Thus, we conclude that mST3Gal V is the fifth-type alpha 2,3-sialyltransferase carrying GM3 synthetic activity.

J Biol Chem, 1999 Jan 8, 274(2), 1092 - 9
Amino acid-dependent control of p70(s6k) . Involvement of tRNA aminoacylation in the regulation; Iiboshi Y et al.; In human T-lymphoblastoid cells, downstream signaling events of mammalian target of rapamycin (mTOR), including the activity of p70(s6k) and phosphorylation of eukaryotic initiation factor 4E-binding protein 1, were dependent on amino acid concentration in the culture media, whereas other growth-related protein kinases were not . Amino acid-induced p70(s6k) activation was completely inhibited by rapamycin but only partially inhibited by wortmannin . Moreover, amino acid concentration similarly affected the p70(s6k) activity, which was dependent on a rapamycin-resistant mutant (S2035I) of mTOR . These data indicate that mTOR is required for amino acid-dependent activation of p70(s6k) . The mechanism by which amino acids regulate p70(s6k) activity was further explored: 1) amino acid alcohols, which inhibit aminoacylation of tRNA by their competitive binding to tRNA synthetases, suppressed p70(s6k) activity; 2) suppression of p70(s6k) by amino acid depletion was blocked by cycloheximide or puromycin, which inhibit utilization of aminoacylated tRNA in cells; and 3) in cells having a temperature-sensitive mutant of histidyl tRNA synthetase, p70(s6k) was suppressed by a transition of cells to a nonpermissible temperature, which was partially restored by addition of high concentrations of histidine . These results indicate that suppression of tRNA aminoacylation is able to inhibit p70(s6k) activity . Deacylated tRNA may be a factor negatively regulating p70(s6k).

Poult Sci, 1998 Dec, 77(12), 1852 - 7
The utilization of dipeptides containing L-arginine by chicken macrophages; Su CL et al.; L-Arginine is the precursor of NO, a cytotoxic agent of macrophages . Studies were carried out to determine whether dipeptides containing arginine can be utilized by lipopolysaccharide (LPS)-activated avian macrophages for NO production . A chicken macrophage cell line, the HD11 cell, was used in all experiments . Peptidase activities were observed in fetal bovine serum (FBS) and macrophage serum free medium (Mac-SFM) . Therefore, the utilization of dipeptides by macrophages was examined using Dulbecco's modified Eagle medium (D-MEM), a chemically defined medium, in short-term culture without FBS . Nitrite accumulation in the culture medium was used as the indicator of NO production . At concentrations of 0.15 mM in the culture media, L-leucinyl-L-arginine was 89% as effective as L-arginine in providing substrate for NO production . L-Argininyl-L-leucine was 38% as effective as L-arginine . The effectiveness increased to 93 and 58%, respectively, when the concentrations of dipeptides and arginine were 1.0 mM . Both values were slightly higher in a second experiment (97 and 70%, respectively) . L-Lysine (10 mM) inhibited nitrite formation from all three sources of L-arginine . In studies of initial rates of transport by HD11 cells in Hanks Balanced Salts solution (HBSS), both L-argininyl-L-leucine and L-leucinyl-L-arginine inhibited arginine uptake . As lysine and arginine share a common transporter for cationic amino acids and are known to compete for transport, these studies suggest that the peptides were hydrolyzed extracellularly, yielding arginine that was transported into the cell where it served as a substrate for NO synthesis.

Infect Immun, 1999 Jan, 67(1), 368 - 74
Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin; Brady CP et al.; Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites . We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults of Schistosoma mansoni and S . japonicum is cathepsin L-like in its substrate specificity . However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2 . We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transforming Saccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1 . Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography . The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec; kcat/Km = 17.25 and 6.24 mM-1 s-1, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L . Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of S . mansoni . Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes . Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases . Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.

Biomaterials, 1998 Nov, 19(21), 1925 - 34
Endothelial cells exposed to erythrocytes under shear stress: an in vitro study; Sirois E et al.; After injury and vascular replacement, endothelial cell recovery is limited and could lead to thrombosis . Seeding small diameter vascular prosthesis with endothelial cells has been proposed to fulfil cell lining and improve surface hemocompatibility . However, detachment of seeded cells occurs following implantation . Previous in vitro studies have looked at the fluid shear stress as a major cause of cell detachment . To our knowledge, the role of erythrocyte collisions has not been investigated . The present in vitro study aims at investigating whether endothelial cell adhesion depends on (i) the presence of erythrocytes in flow and (ii) the latent culture period (1, 24 and 48 h) between seeding and exposure to flow . Endothelial cells were exposed to culture media containing different erythrocyte concentrations using a steady laminar flow of 1350 ml min(-1) in a parallel plate flow chamber . Endothelial cell morphology in dynamic conditions was quantified and compared to that in static conditions . The projected area of cells were mostly found smaller under dynamic than static conditions, particularly at a wall shear stress of 23 dyn cm(-2) . Cells from the 1 h latent culture period were oriented parallel to the flow axis and were more elongated than under static conditions . Conversely, endothelial cell shape was slightly modified when either the latent period or the wall shear stress was increased . Disparate orientation was observed on confluent endothelial cells (24-48 h latent period) exposed to shear stress with or without erythrocytes . Increasing fluid viscous forces due to erythrocytes play a critical role on the behaviour of freshly seeded endothelial cells upon exposure to blood flow.

Med Arh, 1998, 52(3), 143 - 5
Trichomoniasis of the breast diseased by fibrocystic mastopathy: pathogenic rather than saprophytic relationship (Trichomonas in fibrocystic mastopathy process); Krvavac S; Because of the striking similarity between histopathological pictures of chronic trichomonal cervicitis uteri, the tissue reaction after subcutaneous inoculation of Trichomonas culture in experimental animals and female breast diseased by fibrocystic mastopathy, the detection of Trichomonads was undertaken in surgically removed diseased breast parts . In 12 FCM patients, subjected to segmental breast resection, the imprint smears were prepared from dissected specimens and after supravital staining immediately examined by light microscopy . The mucous content from dilated ducts was inoculated in the culture media . The dissected tissues have been further histologically analysed by standard method . In nine out of 12 examined FCM cases the direct microscopy revealed aflagellary, pseudocystic, leucocytoid form of Trichomonads . The cultures were positive in 4 cases: in 3 patients Trichomonas tenax was identified and in the last one T vaginalis . Histopathological findings in all 12 examined cases have shown the changes characteristic for FCM . On the basis of the accumulated knowledge about pathogenic capacity of Trichomonads, it can be with great certainty claimed that these protozoa, even in their pseudocystic form are able to cause the all appearances characteristic for FCM . This first report about Trichomonas infection in the middle of FCM process gives the unexpected hope in solving of its etiology and new insight into antitrichomonal host reaction which is frequently associated with epithelial dysplasia and unrarely with precancerous lesions as earlier observed in cervix uteri.

Atherosclerosis, 1998 Dec, 141(2), 273 - 85
Hepatic lipase mediates an increase in selective uptake of high-density lipoprotein-associated cholesteryl esters by human Hep 3B hepatoma cells in culture; Rinninger F et al.; Selective uptake of high-density lipoprotein- (HDL-) associated cholesteryl esters (CE), i.e . lipid uptake independent from particle uptake, delivers CE to the liver and steroidogenic tissues in vivo . In vitro, besides hepatocytes and steroidogenic cells many other cell types selectively take up HDL CE . Hepatic lipase (HL) stimulates the internalisation of apoprotein (apo) B-containing lipoproteins by hepatocytes independent from lipolysis . In this study the role of HL in the hepatic metabolism of apo A-I-containing lipoproteins, i.e . HDL, was investigated . HDL3 (d = 1.125-1.21 g/ml) was radiolabeled in its protein (125I) and in its CE moiety ({3H}cholesteryl oleyl ether, ({3H}CEt)) . HL originated from tissue culture media of hepatoma cells and from post-heparin plasma . Human Hep 3B hepatoma cells incubated in medium containing radiolabeled HDL3 . In the absence of HL, the rate of apparent HDL3 particle uptake according to the lipid tracer ({3H}CEt) was in most cases in approximately 10-fold excess on that due to the protein label (125I), indicating selective CE uptake from HDL3 . Addition of HL to these incubations increased the cellular uptake of {3H}CEt and of 125I from HDL3 and quantitatively the most prominent effect was an up to approximately 2.5-fold stimulation of apparent selective CE uptake ({3H}CEt-125I) . This increase in selective CE uptake was observed in the presence of tetrahydrolipstatin, an inhibitor of the catalytically active site of HL, suggesting that this HL effect is independent from lipolysis . HL binds to cell surface heparan sulfate proteoglycans . To explore the role of these molecules for the HL effect on selective CE uptake, hepatoma cells were depleted of proteoglycans or Chinese hamster ovary (CHO) cells deficient in proteoglycan synthesis were used . Proteoglycan-deficiency reduced the HL-mediated increase in selective uptake by more than 80% . To investigate if low-density lipoprotein (LDL) receptors or the LDL receptor-related protein (LRP) are involved in the HL effect on selective CE uptake, murine embryonic fibroblasts (MEF) were used which are deficient in these receptors; alternatively, monensin, an inhibitor of endocytosis was present in the medium of Hep 3B cells during the uptake assay for labeled HDL3 . These experiments yielded no evidence for a role of LDL receptors or LRP in the HL-mediated increase in selective CE uptake . In summary, HL mediates an increase in HDL3 selective CE uptake by human Hep 3B hepatoma cells . This HL effect is independent from lipolysis and independent from LRP and LDL receptors . However this HL effect is susceptible to cell surface proteoglycan deficiency . The potential physiologic implication is that HL modifies HDL selective CE uptake by the liver in vivo and such an effect could play a role in reverse cholesterol transport.

Anat Anz, 1998 Dec, 180(6), 511 - 8
Effects of prostaglandin E2 on growth, morphology, morphometry and keratin pattern of bovine corneal epithelial cells cultured in vitro; Parnigotto PP et al.; In this work the relationship between the proliferation of bovine corneal epithelial cells and PGE2 has been studied . Our data indicate that PGE2 plays an important role in the growth of corneal epithelial cells . Actually, epithelial cells cultured on a keratocyte feeder-layer and exposed to indomethacin, a cyclooxygenase inhibitor, have shown a decrease in growth rate at drug concentrations which otherwise did not induce a reduction in the viability of the keratocytes as well as in epithelial cells in separate cultures . This effect has been reversed by an exogenous PGE2 addition to the culture media . Moreover, significant increases have been found in the growth of epithelial cells cultured in the presence of keratocytes, with basal medium and with conditioning medium after adding exogenous PGE2 at concentrations equal to or lower than 10(-6) M . Significant decreases in the dimensions of the corneal epithelial cells have been found only when PGE2 has been added to basal and to conditioning medium, suggesting that the autacoid maintains cell dimension and morphology . The appearance of keratins with high molecular weight (54 and 57 kDa) coupled with the tendency to stratification of the cells cultivated with media supplemented with PGE2, indicates that the autacoid could favour cell differentiation . The action of PGE2 on the corneal epithelial cells does not seem to be influenced by the presence of the fibroblasts and their products, since PGE2 has induced increases in cell growth and morphological variations, independent of cultural conditions and therefore also only in the presence of basal medium.

J Cancer Res Clin Oncol, 1998, 124(11), 598 - 606
Inhibition of tumour cell invasion by protease inhibitors: correlation with the protease profile; Kolkhorst V et al.; The invasive potential of eight established human tumour cell lines of different origin has been studied in the Matrigel assay . Between 25% and 70% of the cells migrated through the Matrigel layer within 24 h, indicating that invasiveness varies with the cell type . Semiquantitative measurements of the proteases MMP-2 and MMP-9, and cathepsins B and L were performed in these cell lines and the cell culture media . High invasive potential was found in those cell lines expressing high levels of cathepsins B and L or matrix metal proteases (MMP), either alone or in combination . Overexpression of one of these enzymes is enough to explain a high invasive potential of a cell line . Selective protease inhibitors at 10 nM concentration in the culture medium were used to inhibit the migration of tumour cells in the Matrigel assay . The MMP inhibitor Batimastat reduced the invasive potential of all cell lines studied independently of the MMP expression . The effect of cysteine protease inhibitors was strongly correlated with the protease profile of the tumour cell line . Our findings support the hypothesis of a very complex activation cascade of matrix-degrading proteolytic enzymes and they underline the need to analyse the protease profile of any tumour before beginning an antiproteolytic tumour treatment.

Ophthalmologe, 1998 Nov, 95(11), 755 - 9
{Effect of E . coli endotoxin on auto-/paracrine function and endothelial cell loss of donor corneas in organ culture}; Sobottka Ventura AC et al.; Corneal cells are known to participate in the regulation of local inflammatory processes by secretion of cytokines . As the corneal endothelium may be exposed to endotoxin in organ culture and endotoxin is known to trigger inflammatory reactions, we investigated the effect of endotoxin from E . coli on organ cultured donor corneae with respect to autocrine and paracrine functions and the endothelial viability and density . 6 pairs of donor corneae were transferred to organ culture . Medium samples were taken prospectively from day 0 to day 20 . On day 10 the medium was changed and one of each pair was incubated with 50 micrograms/ml of endotoxin while the other was immersed in standard organ culture medium . The samples were screened for IL-1, -2, -4, -5, -6, -8, -10, TNF alpha and GM-CSF by ELISA . In addition endothelial cell counts were performed at day 0, after 10 and after 20 days of organ culture, using the fixed frame technique . All endotoxin-incubated organ culture media showed significantly increased IL-6 and -8 levels compared to the fellow cornea and to pre-exposure levels (P < or = 0.004) . In the endotoxin-treated corneae a significantly higher endothelial cell loss occurred (P = 0.007) and signs of degeneration were observed . None of the other cytokines (IL-1 alpha, IL-2, IL-4, IL-5, IL-10, GM-CSF and TNF alpha) were detected during either the first (1 to 10-day) or the second (11 to 20-day) phase of incubation . A correlation seems to exist between an increase in IL-6 and -8 induced by endotoxin, and endothelial cell loss in organ culture.

Biomed Pharmacother, 1998, 52(9), 369 - 77
Cell death induced in lymphocytes expressing the elastin-laminin receptor by excess agonists: necrosis and apoptosis; Peterszegi G et al.; This manuscript summarizes our experiments carried out during the last years on the expression of the elastin-laminin receptor on human activated lymphocytes and cell death triggered by the activation of this receptor by its agonists, elastin peptides . We could distinguish two types of cell reactions, depending on the elastin peptide concentration added to the cell culture media of lymphocytes . At low concentrations (1-10 micrograms/mL, 1.3-13 x 10(-8) M) of kappa-elastin, there was a stimulation of cell proliferation, elastase biosynthesis and release . As the concentration of kappa-elastin was increased in the culture medium up to 100 micrograms/mL, lymphocyte proliferation and elastase production decreased and the proportion of dead cells increased . Cell death was shown to be due to both apoptotic and non-apoptotic mechanisms . Apoptotic cell death increased with agonist concentration and reached approximately 60% of the lymphocyte population at mg/mL elastin peptide concentrations . This observation was confirmed by the concomitant use of several different methodologies, such as flow cytometry and electron microscopy . The precise nature of the non-apoptotic cell death remains to be established.

Eur J Gastroenterol Hepatol, 1998 Jun, 10(6), 497 - 502
The acute phase protein alpha-1-antitrypsin inhibits transferrin uptake in PLC/PRF/5 cells and increases release of hepatitis B virus surface antigen and alpha-fetoprotein; Propst A et al.; BACKGROUND/AIMS: The present study was designed to investigate whether the acute phase protein alpha-1-antitrypsin (alpha1-AT), which has an inhibitory effect on transferrin (tf) receptor-mediated iron uptake in K562 and THP1 cells, has a similar effect in PLC/PRF/5 cells . This hepatic cell line is of specific interest because it is infected with hepatitis B virus (HBV) . Therefore, we addressed the additional question whether alpha1-AT has any effect on cellular protein synthesis and replication of HBV in PLC/PRF/5 cells . METHODS: Cells were incubated with various concentrations of alpha1-AT, dexamethasone, IL-6 and desferrioxamine . HBs-AG, alpha-fetoprotein and albumin concentrations in culture media were measured using commercially available methods . For equilibrium inhibition binding experiments, cells were incubated with 85-182 pmol/l {125I}tf . To study the potential effect of alpha1-AT on DNA synthesis we measured the incorporation of {3H}thymidine into DNA . RESULTS: In equilibrium saturation binding experiments, {125I}tf bound to PLC/PRF/5 cells with K(D) 17.45 +/- 4.57 nM and a maximum density of binding sites of 267,285 +/- 39,915 sites/cell . In inhibition studies alpha1-AT demonstrated an apparently monophasic inhibition of {125I}tf to its receptor . At concentrations > 30 micromol/l alpha1-AT inhibited the growth of PLC/PRF/5 cells up to approximately 50% . The inhibitory effect of alpha1-AT on DNA synthesis was not as potent as that on growth . At the highest concentration of 100 micromol/l, alpha1-AT produced a 35% maximum inhibition of {3H}thymidine incorporation . Incubating PLC/PRF/5 cells with corticosteroids enhanced HBs-AG release significantly . Interestingly, alpha1-AT showed the same pattern of effects on cell metabolism and HBs-AG release as the corticosteroids . When we incubated the cells with 50 micromol/l alpha1-AT, alpha-fetoprotein production increased significantly and HBs-AG release almost doubled . CONCLUSION: We have to assume that there is a specific mechanism inducing HBs-AG release by alpha1-AT, as has been shown to be the case with steroids.

Oncology, 1998 Dec, 55 Suppl 1, 35 - 44
Role of estrogen and estrogen-related growth factor in the mechanism of hormone dependency of endometrial carcinoma cells; Hata H et al.; The role of estrogen and estrogen-related growth factors in the mechanism of hormone dependency of endometrial adenocarcinoma cells was investigated . The proliferation of hormone-responsive human endometrial adenocarcinoma cells (Ishikawa cells), which possess both estrogen and progesterone receptors, was optimally stimulated by 10 nM estradiol . Both transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), added to the culture media, stimulated the proliferation of Ishikawa cells in a dose-dependent manner . Anti-TGF-alpha antibody completely eliminated the stimulatory effects of TGF-alpha . Anti-EGF receptor antibody inhibited the proliferation of these cells . The production of TGF-alpha into culture media was 5-40 pg/10 cells/24 h in 9 human endometrial adenocarcinoma cells . Ten nanomoles of estradiol increased the TGF-alpha production of Ishikawa cells by approximately 2.5-fold of the control level . In contrast, the production of TGF-alpha in hormone-unresponsive HEC-50 cels was not influenced by estradiol . C-erbB-2 oncoprotein expression of human endometrial adenocarcinoma cells, detected by both immunocytochemical staining and Western blot analysis, was associated with the tumor grade of the original tumor tissues . Ten nanomoles of estradiol clearly increased the c-erbB-2 oncoprotein levels at an optimal incubation period of 72 h, whereas estradiol did not affect the expression in HEC-50 cells.

Int J Dev Biol, 1998, 42(7), 833 - 9
Egg culture: the foundation; Hammer RE; Ralph Brinster began his classic work on egg culture more than 35 years ago . His interest in mammalian egg culture had developed, in part, as a consequence of his experiences with animal breeding and reproduction that he gained while growing up on a farm . Ralph decided early in his career that an in vitro approach to culturing eggs would provide a powerful tool with which to study the development of these cells . Beginning at the close of the 19th century, a number of investigators had performed in vitro studies on egg culture and the related area of egg transfer; however, the ability to recover and transplant eggs had reached a much higher level of perfection than had culture . Eggs of many species could be successfully transferred, but there was no reliable technique for egg culture . In 1963, Ralph reported a method for culturing eggs in microdrops of medium under oil (Brinster, 1963), which has become universally used . Two years later, he identified pyruvate as the central and essential energy source for early stages of mouse eggs (Brinster, 1965b) . These two developments revolutionized in vitro studies of mammalian eggs and issued in an era of intense research activity concerning egg culture and egg manipulation . Effective formulations of culture media could now be developed to allow routine in vitro maintenance of eggs, and important parameters for these recipes were soon determined . It was quickly established that the requirement for pyruvate as an energy source exists at ovulation in many species and is already present in germ cells of the mouse fetus . The metabolic activity of the fertilized mouse egg was shown to be low and comparable to bone; however, four days later, at the blastocyst stage of development, the metabolic activity was comparable to brain . Thus, a foundation of understanding about the biology of early mammalian eggs was established between 1960 and 1970, and subsequent studies have broadened this understanding . However, the greatest impact of a simple, reliable egg culture method has been to provide the ability to perform complicated manipulative procedures on preimplantation stages of mammalian embryos . In no area has this been more important than in development of transgenic animals . All methods for generating germ line genetic modifications rely on the ability to maintain and manipulate eggs and early developmental stages in vitro without loss of developmental competence . The importance of efficient egg culture to manipulation and transgenesis is fundamental and enabling.

Am J Clin Nutr, 1998 Dec, 68(6 Suppl), 1505S - 1511S
Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines; Peterson TG et al.; There is substantial variation in the growth inhibition of different human breast cancer cell lines by the isoflavones genistein and biochanin A . ZR-75-1 and BT-20 cells are > or = 2- to 4-fold less sensitive to these isoflavones than are MCF-7 cells, whereas T47D cells have a sensitivity similar to that of MCF-7 cells . To determine whether these differences are related to isoflavone metabolism by these cancer cells, each of the cell lines was incubated with {4-(14)C}genistein and {4-(14)C}biochanin A . Metabolites in the cell culture media were identified by radio-HPLC electrospray ionization mass spectrometry . One metabolite of genistein (genistein 7-sulfate) and 2 metabolites of biochanin A (genistein and genistein 7-sulfate) were detected by radio-HPLC . Further analysis by mass spectrometry identified 3 other metabolites, a hydroxylated methylated form of each isoflavone and a biochanin A sulfate . IC50 (the concentration at which the growth rate was halved) values of the breast cancer cell lines did not correlate well with production of genistein 7-sulfate from genistein or with biochanin A sulfate, genistein 7-sulfate, or genistein from biochanin A . However, IC50 values correlated with the production of the hydroxylated and methylated forms of the isoflavones . Only T47D cells produced these metabolites in this study, and only T47D cells had IC50 values similar to those of MCF-7 cells, which also produced the hydroxylated and methylated metabolites . These data suggest that the hydroxylated and methylated metabolites may be the active forms of genistein in human breast cancer cells and emphasize the importance of isoflavone metabolism in the mechanism of action of isoflavones.

J Virol, 1999 Jan, 73(1), 519 - 32
Herpes simplex virus type 1 vector-mediated expression of nerve growth factor protects dorsal root ganglion neurons from peroxide toxicity; Goins WF et al.; Nerve growth factor beta subunit (beta-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity . Replication-competent (tk- K mutant background) and replication-defective (ICP4(-);tk- S mutant background) vectors were engineered to contain the murine beta-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus . Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of beta-NGF-specific transcripts and beta-NGF protein reaching a maximum at 3 days postinfection (p.i.) . NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells . The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days . In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days . The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures . Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i . whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i . but was ineffective at 3 days p.i . Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of beta-NGF protein expression were detected at 4 wks p.i . only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks . Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active beta-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.

J Pharmacol Toxicol Methods, 1998 Jun, 39(4), 203 - 10
pH drift of "physiological buffers" and culture media used for cell incubation during in vitro studies; Lelong IH et al.; In pharmacological or toxicological studies performed at room atmosphere comparison of various media used for cell incubation revealed discrepancies among results due to pH instability when these media contain bicarbonate . With the classically used protocols, a relatively fast and notable rise of the pH of such media has been observed, and values higher than 8.5 could be reached after 1 h of incubation . A less important rise in pH was also observed for media containing low amounts of sodium bicarbonate, e.g., Hank's formula-derived media . Because Hepes-buffered media or media with abnormal osmolarity cannot always be used for such studies, our choice of media is limited.

Toxicon, 1998 Dec, 36(12), 1807 - 19
Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom; Park D et al.; A venom gland cDNA library of Agkistrodon halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases . Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively . Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al . 1995, J . Biol . Chem . 270, 10246- 10255) . Haly 2 displayed a 78% similarity to beta-fibrinogenase (Hung et al . 1994, B . B . R . C., 205, 1707 1715) . Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein . In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom . Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column . In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasimin . These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.

Tissue Cell, 1998 Oct, 30(5), 525 - 30
Effect of culture conditions on endothelial cell growth and responsiveness; Relou IA et al.; The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium . The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC . The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors . We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic . While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells . Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic . A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation . On non-coated plastic EC were not inducible with bFGF . The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions . The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays . The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation . We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions.

Mediators Inflamm, 1998, 7(2), 73 - 8
Vaccinia virus-regulated acute phase cytokine production in human fibroblasts, U937 cells and endothelium; Rokita H et al.; The production of acute phase cytokines, interleukin 6 (IL-6), tumour necrosis factor (TNFalpha) and interleukin 1 (IL-1beta), was studied in primary cultures of human skin fibroblasts, human monocytic cell line U937 and primary cultures of human umbilical vein endothelial cells (HUVEC) after in vitro infection with vaccinia virus . Significant increase in IL-6 mRNA followed by enhanced protein secretion into the culture media was found in fibroblasts, U937 cells, and HUVEC . TNFalpha increased production in vaccinia virus infected U937 cells resembled closely the pattern of IL-6 production observed in the infected cells . Transient increase in NF-kappaB binding activity was found in the infected U937 (at 90 min) and endothelial (at 30 min) cells . Vaccinia virus induced cytokine production appeared to be transcriptional.

J Immunol Methods, 1998 Oct 1, 219(1-2), 23 - 43
A sandwich type acridinium-linked immunosorbent assay (ALISA) detects soluble ErbB1 (sErbB1) in normal human sera; Baron AT et al.; The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression . In addition to the ErbB1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB1, i.e., sErbB1 . Overexpression of ErbB1 in a variety of tumors has led us to hypothesize that sErbB levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers . To address this hypothesis we have developed an acridinium-linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media . This assay can also detect full-length ErbB1 in cell and tissue extracts . Our ALISA is characterized by high sensitivity (intra-assay LLD < 1 fmol/ml), a broad linear range (approximately 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%) . Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV . Our ALISA does not detect full-length ErbB2, ErbB3, or ErbB4; or p105 soluble ErbB2 . We report that serum sErbB1 levels of healthy women (median = 3716 fmol/ml), ranging in age from 43 to 76 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years . Additional analyses do not indicate that serum sErbB1 levels change with age in either healthy men or women . Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA . Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a approximately 110-kDa protein . We conclude that our ALISA is measuring the relative levels of this p110 sErbB1 analog in normal human sera . Our ALISA, therefore, should be useful for measuring the levels of ErbB1 and sErbB1 molecules in tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.

J Neurobiol, 1998 Nov 15, 37(3), 373 - 82
Gastrin inhibits motility, decreases cell death levels and increases proliferation in human glioblastoma cell lines; De Hauwer C et al.; Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma . This invasion relates mainly to cell motility, which enables individual cell migration to take place . The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs . cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines . A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin . While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines . In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models . All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics.

Morfologiia, 1998, 114(4), 64 - 9
{The direct activating effect of a nonapeptide neurohormone (vasotocin) on the thyroid and interrenal glands of sturgeons in in-vitro experiments}; Platik MM et al.; An in-vitro effect of nonapeptide neurohormone vasotocin on thyroid and interrenal glands was studied in hybrid of Siberian and Lena sturgeons {correction of salmons} at light microscopy level using morphometric method . At a concentration of 0.1 and 1 nmol/l vasotocin was shown to exert undirectional stimulating effect on the thyroid and interrenal gland functions . In the presence of vasotocin at a concentration of 1 nmol/l in culture media the activity of glands is even more pronounced than under the influence of adenohypophyseal hormones, adrenocorticotropic (8 x 10 ng/ml) and thyrotropic (5 ng/ml).

Morfologiia, 1998, 114(4), 41 - 4
{The protective effect of the nerve growth factor in exposing a nerve tissue culture to diphtheria toxin}; Chalisova NI et al.; Diphtheria toxin (1.10(-1)-1.10(-6) Lf/ml) was found to inhibit neurite extension in chick embryo dorsal root ganglia in vitro . If the nerve growth factor (60 ng/ml) was added with toxin in culture media the diphtheria toxin effect was decreased and the neurite outgrowth was compared with control . Protective effect of nerve growth factor by influence of diphtheria toxin may be used in new principles of diphtheria treatment.

Gynecol Oncol, 1998 Nov, 71(2), 177 - 84
Establishment of a new cell line, OKT1, from small cell carcinoma secreting ectopic ACTH of the uterine cervix; Ohtake H et al.; OBJECTIVE: Small cell carcinoma of the uterine cervix is rare and represents a unique entity among gynecological tumors . It sometimes demonstrates neuroendocrine differentiation, including adrenocorticotropin (ACTH) secretion . In this study, we established a new cell line, OKT1, from a case of carcinoma secreting ectopic ACTH without Cushing's syndrome and determined the character of the cell line . METHODS: OKT1 was established from OKT tumor cells, derived from a biopsy specimen of small cell cervical carcinoma, and serially heterotransplanted into nude mice . To characterize OKT1, the cell morphology, growth properties, immunohistochemical properties, hormone- and tumor-associated antigen secretion, tumorigenic potential, DNA profile, and chromosomal alteration were studied . RESULTS: The population doubling time of OKT1 was approximately 27 h . The cytological properties of OKT1, including DNA ploidy pattern, were similar to those of the primary tumor . Neuroendocrine differentiation was shown in the OKT1 cells by the positive immunocytochemical staining of neuron-specific enolase (NSE) and the presence of NSE and ACTH in the culture media . The xenograft of 1 x 10(8) OKT1 cells into nude mice yielded tumor mass . Furthermore, OKT1 demonstrated HPV type 18 and absence of a p53 gene mutation from exons 5 through 8 . CONCLUSION: To our knowledge, OKT1 is the first cell line established from small cell cervical carcinoma with ACTH secretion .

J Chromatogr B Biomed Sci Appl, 1998 Sep 25, 716(1-2), 65 - 75
High-performance liquid chromatographic analysis of biogenic amines in cells and in culture media using on-line dialysis and trace enrichment; Slingerland RJ et al.; A highly sensitive method is presented for the automatic quantitative detection of DOPA metabolites in low concentrations in cells derived from the neural crest using reversed-phase HPLC in combination with fluorescence and electrochemical detection . The HPLC system was combined with on-line dialysis and on-line trace enrichment for the detection of small quantities of DOPA metabolites in culture media . Parameters like detector settings, pH, dialysis time and flow-rates are evaluated and optimized . Static-continuous dialysis can be performed at a low flow-rate concomitant with a high dialysis efficiency (up to >65%) depending on the type of DOPA metabolite . Counterflow dialysis can be used to analyse, with low efficiencies (17-29%), samples consisting of large volumes . Samples containing up to at least 7% (w/v) protein can be analysed in the low flow-rate static-continuous mode . In this last mode of dialysis, limits of detection for dopamine, norepinephrine, epinephrine and n-methyldopamine in DMEM/HAMF12 medium samples are 100 fmol or even lower . Serotonin is detectable at 10 fmol at a signal/noise ratio of 3 . Biogenic amines were detectable at a concentration of 10 fmol/microl in a volume of 100 microl medium with an intra- and inter-assay imprecision <6.4% . This method is applied to study the differentiation level of tumour cells in culture and slices of a tumour derived from the neural crest . With this system, we also detected the excretion of DOPA metabolites from PC-12 cells after treatment with prenylamine.

Hypertension, 1998 Nov, 32(5), 945 - 52
Pressure oscillation regulates human mesangial cell growth and collagen synthesis; Mertens PR et al.; Experimental renal disease models establish glomerular hypertension as a crucial determinant in glomerulosclerosis progression and demonstrate that glomerular capillary pressure reduction delays sclerosis development . An oscillating pressure (OP) chamber was constructed as an in vitro model to study human mesangial cells . Cell cultures were grown under atmospheric pressure (AP) and a controlled OP corresponding to intraglomerular capillary pressure . We show that OP significantly decreases mesangial cell proliferation within 24 hours and attenuates DNA synthesis throughout a 7-day period . To explore the effects of OP on cell metabolism, cell-associated and medium-secreted extracellular (CA and EC, respectively) collagen synthesis were measured by {3H}proline incorporation . In subconfluent cultures, total CA and EC collagen synthesis was unaffected by OP, while in confluent cultures total EC collagen {3H}proline incorporation was increased . To determine whether OP influenced mesangial cell growth induction, the effects of increasing glucose in the cell culture media were investigated . Our data show that the high glucose growth stimulatory effect on cell number and DNA synthesis was suppressed by OP . Under high glucose conditions, total CA collagen synthesis was increased in confluent cultures, whereas the EC collagen fraction remained unchanged . In these cultures, OP caused an additional increase in CA collagen synthesis . This study shows that mesangial cell growth and collagen synthesis are influenced by hyperbaric OP, supporting the hypothesis that glomerular capillary pressure plays a role in progressive glomerulosclerosis development.

Clin Cancer Res, 1996 May, 2(5), 827 - 35
Tumor necrosis factor alpha enhances secretion of transforming growth factor beta2 in MCF-7 breast cancer cells; Danforth DN Jr et al.; We studied the effect of tumor necrosis factor alpha (TNF-alpha) on transforming growth factor beta (TGF-beta) secretion by human breast cell lines to further characterize the antitumor effects of TNF-alpha . We found that TNF-alpha increased the secretion of TGF-beta in two established breast cancer cell lines (MCF-7 and ZR-75-1) but not in two immortalized human mammary epithelial cell lines (184B5 and MCF-10A) . In MCF-7 cells, TNF-alpha increased the secretion of total TGF-beta 6.1-fold within 72 h in a dose-dependent manner . The secretion of both latent and active forms of TGF-beta was increased, and their ratio altered from 25:1 to 12:1 in the medium . TNF-alpha converted the secretory pattern of TGF-beta by MCF-7 cells from the heterodimeric form TGF-beta1.2 to the homodimeric form TGF-beta2 . Immunoblot analysis under nonreducing conditions identified four molecular mass species of TGF-beta secreted in the culture media of untreated MCF-7 cells (238, 210, 40-55, and 25 kDa) . Under reducing conditions, three molecular mass species of TGF-beta were identified: 88, 44, and 12 kDa . Gel filtration analysis demonstrated that the secreted TGF-beta within the range of 12-88 kDa was biologically active . TNF-alpha treatment did not alter the size of molecular mass species secreted by MCF-7 cells and did not change steady-state levels of mRNA for TGF-beta1 or TGF-beta2 . These findings indicate that TNF-alpha may regulate quantitatively and qualitatively TGF-beta secretion by human breast cancer cells in vitro . The diverse biological activities of TGF-beta may also allow TNF-alpha to regulate the growth and metabolism of human mammary epithelial cells and/or stromal cells in a paracrine manner.

Hum Reprod, 1998 Sep, 13(9), 2598 - 601
Effects of interferon-gamma on cytokine production by endometrial stromal cells; Nasu K et al.; To clarify the role of interferon-gamma (IFN-gamma) in reproduction, we have examined its effects on cytokine production by human endometrial stromal cells (ESC) . Concentrations of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage colony-stimulating factor (M-CSF) in the culture media of normal ESC and an endometrial stromal sarcoma cell line, MaMi, were measured using an enzyme-linked immunosorbent assay . Both non-stimulated ESC and non-stimulated MaMi cells constitutively secrete IL-6, IL-8, MCP-1, and M-CSF . In a dose-dependent manner, IFN-gamma increased the concentrations of IL-6, MCP-1, and M-CSF and reduced the concentrations of IL-8 in ESC and MaMi cells . These results suggest that IFN-gamma produced by both decidual inflammatory cells and the developing embryo plays a role in the maintenance of early pregnancy by modulating the production of these cytokines by human ESC.

Rev Cubana Med Trop, 1995, 47(2), 137 - 9
{The use of UIT-A culture medium to produce a biomass for mycobacterial biochemical identification}; Mederos Cuervo LM et al.; The UIT-A solid culture medium is compared with the Lowenstein Jensen, in order to obtain biomass in mounting the "non-tuberculous" mycobacteria biochemical identification test (MNT) with the idea of considering the possibility of using it as a culture medium for mounting and analysing such biochemical tests . Results achieved in both culture media were equal, and that is why the use of UIT-A culture medium is mainly recommended for those strains with a poor or scarce growth.

Fertil Steril, 1998 Oct, 70(4), 647 - 50
Immature oocyte retrieval: lessons from unstimulated IVF cycles; Thornton MH et al.; OBJECTIVE: To describe retrieval of immature oocytes during unstimulated IVF and assess the in vitro maturation and fertilization rates . DESIGN: Retrospective analysis . SETTING: The USC program for assisted reproduction . PATIENT(S): Spontaneously ovulatory women with predominantly pelvic factor as their principal cause of infertility, under the age of 40, and no male factor . INTERVENTION(S): HCG administration in mid-cycle, aspiration of all visible follicles, in vitro maturation of immature oocytes in culture media versus 50% follicular fluid in media, IVF, and embryo transfer . MAIN OUTCOME MEASURE(S): Rates of in vitro maturation, fertilization, and implantation after embryo transfer . RESULT(S): A total of 101 immature oocytes were obtained during 59 follicle aspirations . Thirty percent of prophase I oocytes matured to metaphase II in vitro compared with 44% of metaphase I oocytes . Fertilization rates for matured prophase I oocytes were 62% for those cultured in standard culture media (controls) and 87% for follicular fluid culture media . Two pregnancies resulted from the transfer of embryos derived from immature oocytes when no other embryos were transferred . CONCLUSION(S): Immature oocytes may be retrieved successfully during the mid-cycle aspiration of the dominant follicle in unstimulated IVF cycles . Maturation of immature oocytes in vitro with follicular fluid results in similar maturation and fertilization rates as for control incubation . Immature oocytes thus retrieved contribute to the overall pregnancy success of unstimulated IVF cycles . It may be better to retrieve immature oocytes during unstimulated cycles than during the follicular phase of natural cycles.

J Endocrinol, 1998 Oct, 159(1), 153 - 63
Modulation of skin cell functions by transforming growth factor-beta1 and ACTH after ultraviolet irradiation; Dissanayake NS et al.; The adaptive responses in skin to ultraviolet (UV) radiation include increased cornification of keratinocytes and increased synthesis and distribution of melanin by melanocytes . The possible involvement of paracrine factors in the generation of these responses was studied in a novel two-stage culture model . Human melanocytes or keratinocytes were first irradiated or sham-irradiated and then the conditioned media collected from these cells after 24 h were used to treat unirradiated skin cells . Immunofluorescent staining for transforming growth factor (TGF)-beta1 was increased in UV-irradiated keratinocytes compared with sham-irradiated cells . Increased TGF-beta1 was also detected in the culture media of irradiated keratinocytes . Treatment of unirradiated keratinocytes with conditioned media collected from UV-irradiated keratinocytes resulted in increased absolute numbers and percentages of cornified envelopes per well compared with treatment with conditioned media from sham-irradiated keratinocytes . The magnitude of this effect increased with increased dose of initial irradiation . The effects of conditioned media from UVR-treated cells were mimicked by authentic TGF-beta1 . Treatment of conditioned media from irradiated cells with an antibody shown to neutralise the effects of TGF-beta1 but not with a non-immune antibody of similar isotype, abolished this bioactivity of the conditioned media from UV-irradiated cells . Immunofluorescent staining for ACTH was also increased in UV-irradiated keratinocytes . Conditioned media from UV-irradiated keratinocytes increased tyrosinase activity of unirradiated melanocytes, an effect which was mimicked by authentic ACTH . This bioactivity of conditioned media from irradiated keratinocytes was abolished in the presence of an antibody which neutralised the activity of ACTH but not MSH . These results provide evidence to support the involvement of TGF-beta1 and ACTH in the cornification and pigmentary responses respectively of skin cells after UV exposure.

Environ Health Perspect, 1998 Oct, 106 Suppl 5, 1119 - 24
Characterization of inducible nitric oxide synthase expression in human airway epithelium; Guo FH et al.; Nitric oxide is an important mediator of inflammatory responses in the lung and a key regulator of pulmonary vascular and bronchomotor tone . We have shown that the inducible nitric oxide synthase (iNOS) isoform is continuously expressed in human airway epithelium at mRNA and protein/activity levels in vivo . However, removal of epithelial cells from the in vivo airway environment resulted in rapid loss of iNOS expression, which suggested that expression is dependent upon conditions and/or factors present in the airway . To investigate the mechanisms responsible for maintenance of expression in vivo, we evaluated regulation of iNOS expression in primary human airway epithelial cells . Interferon-gamma (IFN-gamma) was sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and interleukin-4 (IL-4) potentiated the expression through stabilization of iNOS mRNA . The IFN-gamma/IL-4-induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 week . Furthermore, transfer of overlying culture media {conditioned media (CM)} to other HAEC led to iNOS induction . Interestingly, IFN-gamma/IL-4 induction of iNOS was dependent on new protein synthesis, whereas CM induction of iNOS was not . IFN-gamma and IL-4 activated signal transducers and activators of transcription (STAT1 and STAT6) in HAEC, but CM transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines . Thus, IFN-gamma/IL-4, which occurs in human lung lining fluid, led to iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.

Methods Find Exp Clin Pharmacol, 1998 Jul-Aug, 20(6), 451 - 5
In vitro activity of sertaconazole against Malassezia furfur and pachydermatis in different culture media; Palacin C et al.; The activity of sertaconazole against Malassezia furfur and pachydermatis was tested in different culture media in order to elucidate the factors that can influence its activity . The addition to the culture media of lipids, which are necessary for M . furfur to grow, was found to decrease the activity of this antifungal to a great extent . Since the aim of in vitro drug testing is to provide representative data of its activity which must be indicative for its clinical efficacy, and in view of the good clinical response of M . furfur infection to sertaconazole, it is concluded that the activity of this antifungal against M . furfur cannot be determined by the in vitro data provided by conventional methods due to such influential factors.

Cell Motil Cytoskeleton, 1998, 41(2), 126 - 37
Integrin involvement in keratocyte locomotion; de Beus E et al.; Keratocytes are useful in the study of locomotion because they move rapidly (up to 1 micron/second) while maintaining an almost uniform shape, speed and direction . The smooth gliding motion of the keratocyte requires a precise coordination between adhesion, contractility, and retraction . To ask what role integrins play in keratocyte adhesion and locomotion, either RGD peptides or an anti-beta1 integrin mAb that binds to an ectodomain epitope and inhibits adhesion formation was added to the culture media of moving keratocytes . The response to these reagents depended on three interrelated factors: the dose of RGD/mAb, the apparent adhesion strength of the keratocyte to the substratum and the cell speed . High doses cause keratocytes to quickly and irreversibly round up . At intermediate RGD/mAb doses, keratocytes reestablish adhesion after treatment and briefly resume locomotion until partial detachment recurs . At the lowest doses, disruption of beta1 integrin-mediated adhesion formation destabilizes the lamella, temporarily preventing lamellar extension and forward movement of the cell . With increasing culture time, there is an increase in apparent adhesion and a corresponding marked decrease in locomotory velocity . Under these conditions, high doses of RGD/mAb do not cause keratocytes to detach or even produce detectable lamellar instabilities . We postulate that RGD/mAb competitively inhibits new beta1 integrin mediated adhesion formation that is required to support the rates of lamellar extension necessary for rapid locomotion.

Cell Transplant, 1998 Sep-Oct, 7(5), 459 - 68
Comparison of porcine hepatocytes with human hepatoma (C3A) cells for use in a bioartificial liver support system; Wang L et al.; Cells from primary porcine hepatocytes (PPH) and the immortalized human hepatoma cell line C3A are both used in bioartificial liver support systems (BALSS) . In this work the viability and metabolic capacity of PPH and C3A cells cultured in different media were compared . Also, because the cells come into direct or indirect contact with human blood components in BALSS, the effects of human complement on survival and functions of the cells was evaluated . For short-term culture, maintenance of PPH viability was essential for retention of P450IA1 activity (r = 0.882, p < 0.01) and effective ammonia clearance (r = -0.791, p < 0.01) . When cell viability was below 60% P450IA1 activity could not be recorded and nitrogen elimination activity significantly diminished . In contrast to PPH, ammonia levels were markedly increased for C3A cells in all culture media tested (p < 0.01) . Ammonia increase correlated with C3A viability (r = 0.896, p < 0.05) . PPH metabolic function was superior to that of the C3A cell line when evaluated by P450IA1 activity, ammonia removal, and amino acid metabolism . When PPH were incubated in human plasma (HP) or human serum (HS) there was rapid and irreversible deterioration of viability occurring within 9 h . This toxic effect could be prevented by the inactivation of complement . When sodium citrate dissolved in dextrose was added to medium, there was considerable damage to both PPH and the C3A cell line . However, there was no demonstrable toxic effect when hepatic cells of either type were exposed to heparin . We conclude that PPH cultivated in complement-inactivated HP or HS are to be preferred to C3A for clinical application of BALSS, and that heparin should be preferred for anticoagulation in BALSS.

J Assist Reprod Genet, 1998 Sep, 15(8), 455 - 8
Human embryo viability: what determines developmental potential, and can it be assessed?
Gardner DK, Schoolcraft WB.
The understanding of the embryo's nutrient requirements and physiology has led to the development of more physiological culture media, capable of supporting acceptable levels of human blastocyst development in vitro . The success of such media can be attributed to catering to the embryo's changing nutrient requirements, while minimizing culture-induced stress, thereby facilitating normal cell function . Most important, blastocysts derived from such sequential culture systems have a high viability . The ability to identify those blastocysts from within a given cohort which have the highest developmental potential will lead to further increases in implantation and pregnancy rate . Such an approach should ultimately lead to the routine transfer of a single blastocyst in a given IVF cycle, while being able to maintain a high pregnancy rate.

Mol Cell Endocrinol, 1998 Jul 25, 142(1-2), 153 - 63
Ovarian immune cells express granulocyte-macrophage colony-stimulating factor (GM-CSF) during follicular growth and luteinization in gonadotropin-primed immature rodents; Tamura K et al.; To obtain clues as to whether granulocyte-macrophage colony-stimulating factor (GM-CSF) is related to ovarian physiology, the sites, the gene expression and the production of GM-CSF in the ovary during follicular development and luteinization were studied in equine CG (eCG)-primed immature mice and rats . During follicular development, the expression of GM-CSF mRNA was localized in theca-interstitial tissues, oocytes and granulosa cells of small developing follicles in mice . In the mouse ovary after ovulation, luteal tissues as well as the above components had intense signals for GM-CSF mRNA . Mast cells, which were present mainly in the ovarian medulla, also expressed mRNA for GM-CSF in rats . Immunohistochemical analyses with two different antibodies against murine GM-CSF revealed that GM-CSF-like immunoreactivity was detectable mainly in theca-interstitial, luteal tissues, oocytes and mast cells . Intense GM-CSF positive cells in theca-interstitial and luteal tissues were stained with anti-CD11b antibody in mice . Messenger RNAs for GM-CSF receptor subunits were expressed in mast cells of the medulla and in luteal tissues in rat ovary . The levels of GM-CSF released into the culture media by rat ovarian dispersed cells 1-2 days after eCG treatment were higher than those before the treatment, although no significant change in the levels of ovarian GM-CSF mRNA was detected by reverse transcription-polymerase chain reaction analysis . The secretion of GM-CSF was also increased by treatment of the cells with immune stimulators such as phorbol ester, interleukin-1 and lipopolysaccharide . These data indicate that ovarian macrophages and mast cells in addition to theca-interstitial cells, synthesize and release GM-CSF during ovarian cycles, and that ovarian GM-CSF secreting capacity is enhanced during early stages of follicular development in rodents.

Mol Hum Reprod, 1998 Sep, 4(9), 857 - 62
Protein and enzyme patterns in the fluid cavities of the first trimester gestational sac: relevance to the absorptive role of secondary yolk sac; Gulbis B et al.; The potential absorptive role of the yolk sac membrane was evaluated by examining protein and enzyme patterns in embryonic fluids and by comparing the synthetic capacity of the secondary yolk sac, fetal liver and placenta for human chorionic gonadotrophin (HCG) and alpha-fetoprotein (alphaFP) . In yolk sac fluid samples, protein electrophoresis showed two main electrophoretic bands with mobilities comparable to those of albumin and interalbumin-alpha1-globulin, and immunoblotting revealed the presence of albumin, alphaFP, alpha1-antitrypsin, alpha2-macroglobulin, transferrin, complement factors 3 and 4 and immunoglobulin G . In coelomic fluid, similar results were obtained, except for the absence of alpha2-macroglobulin and the presence of ceruloplasmin and IgA . After electrophoresis and immunoblotting with specific antibodies, beta-HCG was detected in all placental homogenates and culture media but was not revealed in any of the corresponding yolk sac tissue samples . Reverse transcription-polymerase chain reaction (RT-PCR) showed that all placental samples express beta-HCG mRNA whereas all yolk sac and liver samples express alphaFP mRNA . These findings suggest that the yolk sac membrane is an important zone of transfer between the extra-embryonic and embryonic compartments and may also help to further develop therapeutic protocols making use of fetal somatic gene therapy by injecting transduced cells into the exocoelomic cavity.

Microbiology, 1998 Sep, 144 ( Pt 9), 2407 - 15
A controllable gene-expression system for the pathogenic fungus Candida glabrata; Nakayama H et al.; A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes . To control the expression of the gene of interest, the C . glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1) . The peptide elongation factor 3 (CgTEF3) and DNA topoisomerase II (CgTOP2) genes from C . glabrata were cloned and their expression assessed using this system . When the promoter of CgTEF3 or CgTOP2 was replaced with tetO::ScHOP1, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth . Repression of the TOP2 or TEF3 gene by doxycycline also hampered the survival of C . glabrata cells in mice; in mouse kidneys the number of C . glabrata cells, in which the TOP2 or TEF3 promoter was replaced with the tetO::ScHOP1 controllable cassette, did not increase when the mice were given doxycycline . Thus, it appears that the gene repression mediated by doxycycline occurred not only in culture media but also in animals; therefore, this system can be used to elucidate the function of the gene in fungal infections and pathogenesis.

J Pediatr Gastroenterol Nutr, 1998 Oct, 27(4), 387 - 92
Selenium deficiency in tissue culture: implications for oxidative metabolism; Baker RD Jr et al.; BACKGROUND: Selenium is located at the catalytic site of the enzyme glutathione peroxidase, and with selenium deficiency the activity of glutathione peroxidase is decreased . Cell culture is an important tool for studying oxidative processes-that is generation and metabolism of oxygen-derived metabolites in the gastrointestinal system . Cell culture is also used to understand the mechanisms of cell injury by oxygen-derived metabolites . METHODS: To assess the importance of the selenium content of cell culture media, Caco-2 cells and the hepatoma-derived cell lines, Hep3B and HepG2, were grown to confluence and placed in media with various concentrations of selenium . After 7 to 14 days, cells were harvested and assayed for glutathione peroxidase, lactate dehydrogenase, and protein content . RESULTS: Cells maintained in media unsupplemented with selenium demonstrated a progressive decrease in glutathione peroxidase activity . Cells maintained in media supplemented with various concentrations of selenium demonstrated a dose-dependent increase in glutathione peroxidase until a plateau was reached . The plateau was reached at approximately 400 times the selenium concentration routinely used in cell culture . In the Caco-2 and hepatoma cells, no toxicity was observed at selenium supplementation five times the lowest concentration needed to reach a plateau . CONCLUSIONS: Cell culture media are routinely deficient in selenium, and cells that are cultured in this medium are deficient in glutathione peroxidase activity . Studies of oxidative metabolism based on cultures deficient in selenium may yield results that could be falsely interpreted . The addition of 1 nM selenium is sufficient for these cell lines to reach a plateau for intracellular glutathione peroxidase activity . These observations may have important ramifications for the study of reactive oxygen metabolite injury in cell culture.

Exp Eye Res, 1998 Sep, 67(3), 323 - 9
Sulfation of intrinsic glycoproteins of the rabbit vitreous; Goes RM et al.; The experiments reported here were designed to characterize the intrinsic vitreous glycoproteins and to understand the process of their sulfation . Rabbits were injected intravitreally with 35S-sodium sulfate and killed at several time intervals after injection . In another series of experiments, rabbits were injected either with 35S-sodium sulfate, 3H-fucose or 3H-tyrosine, associated or not associated with tunicamycin administration . Vitreous from the control eyes was also digested with N-glycosidase . Furthermore, ciliary bodies, the putative source of the intrinsic vitreous glycoproteins, were incubated with 35S-sodium sulfate in the presence or absence of the protein synthesis inhibitor cycloheximide, and the culture media recovered for analysis . These and the vitreous samples of the other experiments were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography . Except for serum albumin, practically all polypeptide bands of the vitreous and culture media were labeled with radioactive sulfate and were shown to undergo renewal . The experiments using tunicamycin or enzyme treatment suggest that radioactive sulfate was incorporated not only into the carbohydrate side chains of the glycoproteins but also into the amino acid tyrosine of the polypeptide backbone of these glycoproteins .

J Biol Chem, 1998 Oct 23, 273(43), 28208 - 18
Molecular cloning of the oncofetal isoform of the human pancreatic bile salt-dependent lipase; Pasqualini E et al.; Specific transcripts for bile salt-dependent lipase (BSDL), a 100-kDa glycoprotein secreted by the human pancreas, were immunodetected in BxPC-3 and SOJ-6 pancreatic tumoral cell lines . Sequencing of fragments, obtained by mRNA reverse transcription and amplification, confirmed the presence of BSDL transcripts in these cancer cells . The protein was detected in lysates of pancreatic tumoral cells, where it was mainly associated with membranes . Only a minute amount of the enzyme was detected in the culture media . Immunofluorescence studies demonstrated that in SOJ-6 cells, BSDL colocates with the p58 Golgi protein and suggested that the protein may be sequestrated within the Golgi compartment . These results demonstrated that BSDL is expressed in human pancreatic tumoral cells and cannot be secreted (or for the least very poorly) . Subsequently, a cDNA covering the entire sequence of BSDL was obtained by reverse transcription-polymerase chain reaction . The sequence of this cDNA indicated that the N-terminal domain encoded by exons 1-10 was identical to that of BSDL expressed by the human normal pancreas . However, the sequence corresponding to exon 11, which should code for the 16 tandem-repeated identical mucin-like sequences of BSDL, was deleted by 330 base pairs (bp) and encoded only 6 of these repeated sequences . We conclude that this truncated variant of BSDL would be its oncofetal form, referred to as feto-acinar pancreatic protein . We then investigated whether the deletion of 330 bp affected the secretion of the protein . For this purpose, the cDNA corresponding to the mature form of the BSDL variant expressed in SOJ-6 cells was cloned into an expression/secretion vector and transfected into CHO-K1 cells . Results indicated that the variant of BSDL isolated from SOJ-6 cells was expressed and secreted by transfected cells . However, the level of BSDL secreted by these transfected CHO-K1 cells was significantly higher than that observed for SOJ-6 cells . Consequently, the retention of the oncofetal variant of BSDL observed in human pancreatic tumoral cells might not result from inherent properties of the protein.

Sex Transm Dis, 1998 Sep, 25(8), 418 - 20
False-positive enzyme immunoassay test results for Chlamydia trachomatis because of contact of the collection swab with agar; Sales V et al.; BACKGROUND: We noted an increased incidence of false-positive Chlamydia trachomatis enzyme immunoassay results using the Abbott IMx SELECT C . trachomatis EIA test when a single swab was used for urethral sampling for both gonococcal culture inoculation and chlamydial detection . GOALS: To evaluate if contact of the enzyme immunoassay collection swab with an agar produces false-positive chlamydial enzyme immunoassay results . STUDY DESIGN: Samples containing agar-based culture media were tested by two enzyme immunoassays and a ligase chain reaction technique . RESULTS: We report false-positive chlamydial enzyme immunoassay results using the Abbott IMx SELECT C . trachomatis EIA test if the collection swabs are in contact with gonococcal culture media (Modified New York City agar, chocolate, Thayer-Martin, or GC-lect) before insertion of the swab in the transport media of the enzyme immunoassay . The other assay results were negative . CONCLUSIONS: Using a single collection swab to screen for genital infections with gonococcal cultures and chlamydial enzyme immunoassay is inappropriate because it may lead to false-positive chlamydial enzyme immunoassay results, at least with the Abbott IMx SELECT C . trachomatis EIA test, incurring public health and financial consequences.

J Clin Endocrinol Metab, 1998 Oct, 83(10), 3609 - 14
Augmentation of leptin synthesis and secretion through activation of protein kinases A and C in cultured human trophoblastic cells; Yura S et al.; Leptin is a fat cell-derived hormone that regulates food intake and energy expenditure . We previously demonstrated that leptin is produced by nonadipose cells, i.e . by placental trophoblasts . We also reported that a human trophoblastic cell line, BeWo cells, expresses leptin gene and secretes leptin into culture media . To elucidate the regulatory mechanisms of leptin production by human trophoblasts, we investigated synthesis and secretion of leptin in BeWo cells and in explant cultures of human placental tissue . Leptin production and gene expression in BeWo cells were increased by treatment with forskolin . The forskolin-induced increase in leptin production was completely suppressed by H89, an inhibitor of protein kinase A . Leptin production and gene expression in BeWo cells were increased by treatment with phorbol myristate acetate (PMA) . The PMA-induced increase in leptin production was completely suppressed by H7 and staurosporine, both of which are inhibitors of protein kinase C . Leptin secretion from first trimester chorionic tissue was approximately 50-fold greater than that from term placental tissue . Leptin production and gene expression in explant cultures of placental tissue at both stages of pregnancy were augmented markedly by treatment with forskolin or PMA . The present study demonstrated augmentation of leptin production by protein kinase A and protein kinase C in cultured human trophoblasts, thereby leading to a better understanding of the regulatory mechanisms of leptin production in human trophoblasts in vivo.

Rev Cubana Med Trop, 1996, 48(1), 40 - 4
{New solid culture media for growing Borrelia persica and Borrelia microti}; Bahrmand AR et al.; A new solid means for the fast detection of Borrelia persica and Borrelia microti is described . Generally, culture and isolation of Borrelia takes about 21 days . The serological test, which is carried out more often, takes less time but it is associated with false positive reactions relatively high . However, our new solid means reduces the culture time to 72 hours, allowing to have a fast diagnosis of the disease caused by Borrelia persica and Borrelia microti, and to start the early treatment of these patients.

Rev Cubana Med Trop, 1992, 44(3), 181 - 4
{Obtaining a clone of the AP-61 cell line . Its usefulness in the multiplication of dengue viruses 1 and 2}; Morier Diaz L et al.; The obtention of a clone of the cell line AP-61 (Aedes pseudoscutellaris) is reported . Details of the cloning and culture media employed are discussed . Usefulness of the clone for the multiplication of dengue 1 and 2 viruses is proved in comparison with the original line using the indirect immunofluorescence technique.

Am J Respir Cell Mol Biol, 1998 Oct, 19(4), 629 - 35
Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells; Yao XL et al.; Clara cell secretory protein (CCSP), or CC10, is an inhibitor of secretory phospholipase A2 which may be produced by phagocytic cells and by a variety of other cells in the airway . Tumor necrosis factor-alpha (TNF-alpha) is capable of activating phospholipases and inducing the expression of a variety of genes in the airway epithelium which may modulate the airway inflammatory response . Therefore, it was of interest to determine whether this proinflammatory cytokine could induce the production of a counterregulatory protein such as CCSP which might modulate the production of eicosanoid mediators in the airway . Using a human bronchial epithelial cell line (BEAS-2B), CCSP messenger RNA (mRNA) levels were detected by ribonuclease protection assay . TNF treatment of these cells increased CCSP mRNA levels in a time- and dose-dependent manner . The CCSP mRNA level increased in response to TNF-alpha (20 ng/ml) stimulation after 8 to 36 h with the peak increase at 18 h . Immunoblotting of CCSP protein released into the culture media demonstrated that TNF-alpha induced the synthesis and secretion of CCSP protein in a time-dependent manner over 8 to 18 h . The results of a CCSP reporter gene activity assay, nuclear run-on assay, and CCSP mRNA half-life assay indicated that the TNF-alpha-induced increases in CCSP gene expression are regulated at the post-transcriptional level . We conclude that TNF-alpha induces airway epithelial cell expression of human CCSP protein and may modulate airway inflammatory responses in this manner.

Appl Environ Microbiol, 1998 Oct, 64(10), 3900 - 9
Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry
Robertson BR, Button DK, Koch AL.
The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory . A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment . It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation . Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve . When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density . This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream . Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4', 6-diamidino-2-phenylindole) fluorescence intensity . The total biomass of the mixture was 340 &mgr;g/liter . The lowest value for mean dry mass, 0.027 +/- 0.008 pg/cell, was for the subpopulation of C . oligotrophus containing cells with a single chromosome . Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli . The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10(3) cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

Am J Physiol, 1998 Oct, 275(4 Pt 2), R976 - 85
Leptin does not fully account for the satiety activity of adipose tissue-conditioned medium; Weigle DS et al.; To determine whether leptin alone accounts for the satiety activity secreted by native adipose tissue, we prepared culture media conditioned by microdissected adipose tissue from overfed Long-Evans rats, fa/fa rats, or db/db mice (media A, B, and C, respectively) . Medium A significantly suppressed food intake following intracerebroventricular delivery to Long-Evans rats (2-h chow intake = 68 +/- 5% of baseline, P < 0.001) . Media B and C significantly suppressed food intake following intraperitoneal delivery to ob/ob mice (24-h chow intake = 56 +/- 7% of baseline for medium B, P = 0 . 001; 4-day chow intake = 78 +/- 3% of baseline for medium C, P = 0 . 004) . Using a leptin receptor-based bioassay, we determined that the leptin concentration of medium C was 392 +/- 18 ng/ml . This concentration was 20-fold lower than the concentration of recombinant murine leptin required to produce a similar degree of feeding suppression following 5 days of administration to ob/ob mice . Neither medium conditioned by adipose tissue from ob/ob mice nor medium conditioned by adipose tissue from fa/fa rats and subsequently immunodepleted of leptin had significant satiety activity . We conclude that leptin is necessary but not sufficient to account for the satiety activity of native adipose tissue, perhaps due to the production by adipocytes of a cofactor that augments the ability of leptin to suppress feeding.

Neurosci Lett, 1998 Aug 7, 252(1), 17 - 20
Anti-gamma interferon can prevent the premature death of trisomy 16 mouse cortical neurons in culture; Hallam DM et al.; Previous reports have indicated that human trisomy 21 and mouse trisomy 16 neurons exhibit decreased viability in culture when compared to euploid control cultures and that trisomic cells are significantly more sensitive to the anti-cellular effects of the interferons . In the study reported here, cortical neurons from euploid and trisomy 16 mouse fetuses were treated with either anti-gamma-interferon or non-specific IgG and neuron morphology and viability measured photographically . The addition of anti-gamma-interferon IgG to the culture media had no effect on euploid neurons, but significantly increased trisomy neuron viability throughout the 5-day culture period . Assay of both DNA fragmentation and phosphatidylserine externalization suggested that the trisomic neurons were undergoing apoptosis at a significantly higher rate than their euploid counterparts and that this increase in apoptosis could be almost completely prevented by addition of either ligand purified monoclonal or ligand purified polyclonal anti-gamma-interferon IgG . Taken together, these data suggest that endogenous interferon plays an important role in the premature death of the trisomy neuron.

Hum Reprod, 1998 Aug, 13(8), 2234 - 7
Ultrasound covers and sonographic gels are embryo-toxic and could be replaced by non-toxic polyethylene bags and paraffin oil; Van der Auwera I et al.; The objective of this study was to test the hypothesis that ultrasound covers and sonographic gels, used during vaginal ultrasound, are toxic for mouse embryonic development in vitro . A prospective randomized design was used on pronucleate ova of F1 hybrid CBA x C57Bl female mice . The mice were superovulated with pregnant mare's serum gonadotrophin and human chorionic gonadotrophin and mated with CBA x C57Bl males . The pronucleate ova were randomly divided between culture media with the addition of commercially available ultrasound covers and sonographic gels in different concentrations . As controls and potential alternatives, plastic polyethylene bags and paraffin oil were tested simultaneously . Embryo-toxicity was assessed by documenting cleavage capacity, blastocyst formation and embryo degeneration in vitro . Exposure of culture medium to the ultrasound covers and sonographic gels tested resulted in a severely reduced cleavage capacity, a high incidence of embryo degeneration and absent or impaired blastocyst formation . This toxic effect could be reduced by high dilutions in vitro . In contrast, plastic polyethylene bags and paraffin oil had no influence on in-vitro development of mouse ova . We conclude that commercially available ultrasound latex covers and sonographic gels are toxic for mouse embryos and can potentially influence embryonic development during infertility treatment . It is safer to perform vaginal ultrasonic measurements using non-toxic paraffin oil (as contact fluid) and plastic polyethylene bags (as ultrasonic cover).

Alcohol Clin Exp Res, 1998 Sep, 22(6), 1245 - 54
The effects of chronic ethanol consumption on the formation of phosphatidylethanolamine molecular species and their appearance at the plasma membrane; Seenaiah B et al.; The purpose of our study was to determine whether chronic ethanol consumption affected membrane assembly by altering the formation of specific molecular species of phosphatidylethanolamine (PE) and their subsequent incorporation into the plasma membrane (PM) . We investigated the effects on the PE species made by the two major pathways in hepatocytes: (1) from CDP-ethanolamine in the endoplasmic reticulum, and (2) by the decarboxylation of phosphatidylserine (PS) in the mitochondria . Ethanol consumption exerted significant effects on the formation of ethanolamine-derived PE species and affected mainly two species, the 16:0/22:6 and 18:0/20:4 species . In cultured hepatocytes from ethanol-fed rats labeled with {3H}ethanolamine for 0.25 to 4 hr, the amount of the {3H}16:0/22:6 PE species was decreased compared with that in control cells, whereas the amount of {3H}18:0/20:4 species was increased . The amount of the {3H}16:0/22:6 PE species on the cell surface was also decreased in hepatocytes from ethanol-fed rats, whereas the amount of {3H}18:0/20:4 species was increased . In contrast, the profile of {3H}PE species formed in cells treated with {3H}serine exhibited minor alterations, and the profile of the serine-derived {3H}PE species on the cells surface was not altered after 4 hr of labeling . The changes in ethanolamine-derived species were apparently caused by time-dependent alterations in the metabolic processes, because the presence of 110 mM ethanol in the culture media did not affect the profiles of {3H}PE species in cells from control or ethanol-fed rats and was not required to sustain the altered profiles . The results indicate that the synthesis of specific PE molecular species and their appearance on the PM may occur by compartmentalized processes which are distinguishable by different sensitivities to ethanol consumption . The results indicate that ethanol consumption may contribute alcoholic hepatic injury by interfering with the metabolism of specific PE molecular species and their assembly into the PM.

Hum Reprod, 1998 Jun, 13 Suppl 3, 148 - 59; discussion 160
Culture of viable human blastocysts in defined sequential serum-free media; Gardner DK et al.; In human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development, resulting in a 10-15% implantation rate . However, in other mammalian species, the transfer of cleavage stage embryos, which normally reside in the oviduct, to the uterus results in a significantly lower implantation rate compared with blastocysts . It is therefore proposed that, in order to increase implantation rates in human IVF, one has to move to extended culture and transfer at the blastocyst stage . The transfer of blastocysts will not only help synchronize the embryo with the female tract but will facilitate the identification of those embryos with little or no developmental potential . In order to culture viable blastocysts it is important to use more than one culture medium to cater for the changing requirements of the preimplantation embryo as it develops and differentiates . If sequential culture media are not used, one can obtain blastocysts but their resultant viability is low . The use of sequential serum-free media in human IVF has resulted in > 50% of embryos becoming blastocysts with an implantation rate of approximately 50% . Further advances in human embryo culture should come from the replacement of protein with the glycosaminoglycan hyaluronate, which is more suitable than albumin in supporting implantation in the mouse, and which will eliminate biological variation and possible contamination from blood products . With the routine culture of human blastocysts will come the introduction of non-invasive tests of embryo viability, capable of identifying those blastocysts most likely to develop from a given cohort . As the implantation rate of blastocysts is higher than that of the cleavage stage embryo, fewer embryos will be required for transfer in order to establish a successful pregnancy, thereby reducing the number of multiple gestations and increasing the overall efficiency of human IVF.

Hum Reprod, 1998 Jun, 13 Suppl 3, 137 - 44; discussion 145-7
Culture and quality control of embryos; Cohen J et al.; Improvement of embryo quality during in-vitro culture can be achieved by understanding and controlling the requirements of gametes and embryos . The most obvious route is to alter culture media, but standardization could be influenced by diverse environmental factors . Abnormal embryos from patients with multiple failures probably do not benefit from standardization and require specialized therapy, that is if their physiology is not already irreversibly jeopardized during gametogenesis . This paper describes the adverse environmental factors present in laboratory air and released by common products used by laboratories . Assays and results of the air determinations in several laboratories are reported, as well as potential counter measures . The possibility of altering the immediate environment of the nucleus of the egg by ooplasmic transplantation is also considered, and the first attempts resulting in two ongoing pregnancies are reported.

Proc Soc Exp Biol Med, 1998 Oct, 219(1), 64 - 8
Chelation of extracellular zinc inhibits proliferation in 3T3 cells independent of insulin-like growth factor-I receptor expression; Thornton WH Jr et al.; Depletion of zinc inhibits growth in animals and proliferation of cultured cells . Additionally, zinc can serve as an antioxidant protecting many compounds, including proteins, from oxidation . Regulation of cell division also involves insulin-like growth factor type I (IGF-I) and its receptor, especially during late G1 phase, allowing progression of the cell to S phase with subsequent DNA synthesis . We examined the effects of zinc depletion from the culture media of Swiss 3T3 cells on the cell cycle and IGF-I receptor expression . Cells were exposed to reduced fetal bovine serum concentrations to induce growth arrest, then returned to normal fetal bovine serum concentrations with the divalent cation chelator diethylenetriamine pentaacetic acid . Reducing the fetal bovine serum concentration did not induce quiescence in the cells as previously suggested . Zinc depletion reduced the proliferative fraction (S and G2/M phases) of the cell cycle . The addition of glutathione to the zinc-depleted media partially returned the proliferative fraction to the control level . Fetal bovine serum deprivation reduced IGF-I receptor expression whereas the absence of zinc had little effect on receptor expression . We conclude that depletion of zinc from culture media inhibits 3T3 cell proliferation independent of insulin-like growth factor-I receptor expression, and part of this inhibition is due to the antioxidant capacity of this divalent cation.

Lett Appl Microbiol, 1998 Aug, 27(2), 106 - 10
Comparison of conventional culture and PCR methods for the detection of Legionella pneumophila in water; Villari P et al.; A comparative assessment of conventional culture and nucleic acid techniques in the detection of Legionella pneumophila in seeded tap water samples was performed, using bacterial concentrations ranging from 994 to 0.015 cfu ml-1 . Different filtration and centrifugation protocols were evaluated . The results permitted the development of a tentative algorithm for the detection of legionellae in tap water . Samples should first be analysed using PCR methods . In the event of quantitative data and bacterial strains for epidemiologic typing being required, the same sample, or a greater volume of the sample, if positive with PCR, can be re-tested by filtration through polycarbonate membranes followed by plating a homogenate of the filter . If samples are found to be negative with PCR, they can be re-analysed in greater volumes by filtration through polycarbonate membranes followed by direct placing of the filter on culture media, to allow detection of very low numbers of bacteria . This protocol should be validated in the field before it can be routinely implemented.

Matrix Biol, 1998 Aug, 17(4), 255 - 68
Structural analysis of proteoglycans synthesized by mineralizing bone cells in vitro in the presence of fluoride; Waddington RJ et al.; This study investigated the biochemical structure of proteoglycans synthesized during matrix maturation by mineralizing bone cells in vitro, in the presence and absence of fluoride . Bone cells were obtained from rat femur washes and cultured in alpha MEM media supplemented with fetal calf serum, ascorbic acid, beta-glycerophosphate and dexamethasone . Cells were characterized as osteoblast-like by the expression of alkaline phosphatase activity and the synthesis of collagen type I and osteocalcin . Fluoride, present in the culture media at concentrations of 10(-5) M or 10(-7) M, had negligible effect on cell viability . However, calcium deposition was increased in cell cultures incubated in the presence of fluoride . Proteoglycans were extracted from the extracellular matrix with 4 M guanidinium chloride and purified by anion exchange chromatography . Biochemical analysis identified the presence of the small leucine rich proteoglycan, decorin and biglycan, in addition to degradation products relating to the larger chondroitin sulphate protoeglycan, versican . Fluoride had little effect on the size or amino acid composition of the protein core, but resulted in significant alterations to the GAG chains, including a dramatic reduction in chain length, reduction in sulphation and decrease in the proportion of dermatan sulphate compared to chondroitin sulphate . The influence of fluoride on proteoglycan structure synthesized by mineralizing bone cells provides valuable information, indicating specific roles for dermatan sulphate and chondroitin sulphate proteoglycans . The results suggested that fluoride affected the post-translational assembly of the GAG chains which may be an influential factor in the mineralization process.

J Biol Chem, 1998 Oct 2, 273(40), 25937 - 43
Two forms of collagen XVII in keratinocytes . A full-length transmembrane protein and a soluble ectodomain; Schacke H et al.; The cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet . Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells . Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa alpha1(XVII) chains . The globular intracellular domain was disulfide-linked, and the N-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures . A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain . The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides . Northern blot analysis with probes spanning either the distal 5'or the distal 3' end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally . In concert, keratinocytes harboring a homozygous nonsense mutation in the COL17A1 gene synthesized neither the 180-kDa alpha1(XVII) chain nor the 120-kDa polypeptide . Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide . These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing . Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain.

Biol Reprod, 1998 Oct, 59(4), 1008 - 15
Estrogen and lipopolysaccharide stimulation of prostacyclin production and the levels of cyclooxygenase and nitric oxide synthase in ovine uterine arteries; Vagnoni KE et al.; Several enzymes play a role in vasodilation, including cyclooxygenase, which converts arachidonic acid into prostaglandins, and nitric oxide synthase, which converts arginine to citrulline and yields nitric oxide . The effects of endogenous and exogenous estrogen and lipopolysaccharide on uterine artery production of prostacyclin, and levels of cyclooxygenase and nitric oxide synthase were examined . Uterine arteries collected from ewes during the follicular (Day -1 to 0, Day 0 = estrus) or luteal (Day 10) phase were treated in vitro with lipopolysaccharide . In addition, ovariectomized ewes were treated in vivo with estradiol-17beta (5 microg/kg; 120 min) or a vehicle control; arteries from the uteri were treated in vitro with lipopolysaccharide . After 24 h of lipopolysaccharide treatment, culture media were collected for measurement of 6-keto-prostaglandin F1alpha (the stable metabolite of prostacyclin) . These uterine arteries were homogenized, and the level of cyclooxygenase and nitric oxide synthase was determined by Western analysis . Lipopolysaccharide stimulated (p < 0.02) prostacyclin production by uterine arteries from both follicular- and luteal-phase sheep although phase of the estrous cycle did not affect prostacyclin responses (p = 0.56) to lipopolysaccharide . In contrast, uterine arteries from ovariectomized sheep treated with estradiol-17beta produced more prostacyclin (p < 0.001) in response to lipopolysaccharide than did uterine arteries from ovariectomized sheep treated with the vehicle control . There was no effect of phase (follicular or luteal) of the estrous cycle on either cyclooxygenase-1 or -2 gene expression . Lipopolysaccharide increased (p = 0.0002) gene expression of cyclooxygenase-2, but not cyclooxygenase-1, in both follicular- and luteal-phase ewes, which was significantly correlated (r2 = 0.91, p = 0.003) with uterine artery production of prostacyclin . Uterine arteries from follicular-phase sheep expressed significantly more nitric oxide synthase-III after lipopolysaccharide exposure than did uterine arteries from luteal-phase ewes (p = 0.03) . In contrast, nitric oxide synthase-II was not detected in uterine arteries after lipopolysaccharide exposure . These results suggest that estrogen plays a role in regulating uterine artery responses to lipopolysaccharide.

Biol Reprod, 1998 Oct, 59(4), 969 - 77
Transient expression of a translation initiation factor is conservatively associated with embryonic gene activation in murine and bovine embryos; De Sousa PA et al.; In the present study the abundance of mRNAs for eukaryotic translation initiation factors eIF-1A (formerly known as eIF-4C), -2alpha, -4A, -4E, and -5 was examined in in vivo-derived mouse embryos throughout preimplantation development using a semiquantitative reverse transcription-polymerase chain reaction assay . Although the mRNA profile for each gene is unique, only mRNA for eIF-1A transiently increases during embryonic gene activation (EGA) at the 2-cell stage, and this was confirmed by an independent hybridization-based assay . In in vitro-developed bovine embryos, mRNA for eIF-1A was transiently detected at the 8-cell stage, when the major activation of the genome occurs in this species . As in the mouse, detection in 8-cell bovine embryos was sensitive to the transcriptional inhibitor alpha-amanitin . It was also observed at the same time relative to cleavage in embryos cultured in defined medium under a reduced oxygen environment, and in medium supplemented with serum and somatic cells in 5% CO2 in air . Neither the chronology of early cleavage divisions nor the yield of bovine blastocysts differed in these culture media . Our results suggest that transient expression of eIF-1A in the mouse and cow is a conserved pattern of gene expression associated with EGA in mammals.

Infect Immun, 1998 Oct, 66(10), 4976 - 80
Plasminogen binding and activation at the surface of Helicobacter pylori CCUG 17874; Pantzar M et al.; The binding of iodine-labelled plasminogen to Helicobacter pylori CCUG 17874 was characterized . Inhibition of the binding was observed after preincubation of H . pylori cells with nonradiolabelled plasminogen, lysine, or the lysine analogue epsilon-aminocaproic acid . Fragments of plasminogen, kringles 1 to 3, kringle 4, and mini-plasminogen, were also studied as potential inhibitors . Mini-plasminogen caused total inhibition of the plasminogen binding, while the other fragments caused only partial inhibition . These findings suggest that H . pylori binds specifically the fifth kringle structure of the plasminogen molecule . Plasminogen binding to H . pylori seems to be independent of culture media and independent of the presence of the cytotoxin-associated CagA antigen . Immunoblot analysis identified two plasminogen binding proteins of 57 and 42 kDa . Scatchard plot analysis revealed one binding mechanism with a Kd value of 7 x 10(-7) M . Conversion of H . pylori cell-bound plasminogen to plasmin in the presence of a tissue-type plasminogen activator was demonstrated by digestion of the chromogenic substrate S-2251 . No activation was noted when plasminogen or tissue-type plasminogen activator was incubated with H . pylori cells alone . Formation of H . pylori cell surface-bound plasmin may be important to provide a powerful proteolytic mechanism for gastric tissue penetration in type B gastritis and peptic ulcer disease, since plasmin degrades not only fibrin but also extracellular matrix proteins such as various collagens and fibronectin.

J Control Release, 1998 Apr 30, 53(1-3), 289 - 99
Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids; Pouton CW et al.; DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture . Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups . The particle sizes and zeta potentials of a range of complexes were determined . Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media . The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter . Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection . Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine) . Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments . Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine . In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency . There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency . In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture . As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity . The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.

J Control Release, 1998 Apr 30, 53(1-3), 137 - 43
Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides; Fattal E et al.; Antisense oligonucleotides with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene . However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration . In view of using oligonucleotides as active molecules, the development of polymeric particulate carriers was considered . Oligonucleotides were associated with biodegradable polyalkylcyanoacrylate nanoparticles through the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations . Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of the capture of nanoparticles by an endocytotic/phagocytotic pathway . The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice and the stability of these molecules has been evaluated by original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by multichannel radioactivity counting . Stability in vivo in the plasma and in the liver was shown to be improved when the oligonucleotides were adsorbed onto the nanoparticles . These results obtained both in vitro and in vivo open exciting perspectives for the specific delivery of oligonucleotides to the liver, thus considering this approach for the treatment of liver diseases (e.g . liver metastasis or hepatitis).

Endocrine, 1998 Jun, 8(3), 261 - 7
Regulation of prostaglandin F2alpha-receptor mRNA in human granulosa-luteal cells by human chorionic gonadotrophin and prostaglandin F2alpha; Vaananen JE et al.; This study examined the effects of prostaglandin F2alpha (PGF2alpha) and human chorionic gonadotropin (hCG) on the levels of PGF2alpha-receptor (PGF2alpha-R) mRNA and steroidogenesis, in the human granulosa luteal cell (hGLC) . Human GLCs collected from patients undergoing in vitro fertilization, were cultured for 24 h, after which cells were exposed to culture media containing either vehicle, hCG (1IU/mL), or hCG plus PGF2alpha (10(-11)-10(-6) M), for a further 24 h . Following the treatment period, media were collected and stored (-20 degrees C) until assayed for progesterone and 17beta-estradiol (estradiol) . Immediately following the treatment period, cells were extracted for total RNA . Transcripts for PGF2alpha-R were detected by PCR with two different sets of oligonucleotide primers based on the published human and rat PGF2alpha-R sequences . PCR products were confirmed to be those of PGF2alpha-R by size and by Southern blot hybridization with an internal oligo nucleotide probe . All experiments were performed a minimum of three times, on cells from a minimum of three separate patients . Prostaglandin F2alpha-R mRNA was significantly downregulated, whereas progesterone and estradiol production were significantly stimulated by hCG . Conversely, both low (10(-11)M) and high concentrations (10(-6) M) of PGF2alpha restored PGF2alpha-R mRNA levels to those of the controls, whereas steroidogenesis was significantly inhibited by these conditions . At a concentration of 10(-9)M PGF2alpha-R mRNA was barely detectable . Progesterone and estradiol production were inversely related to PGF2alpha-R levels, since hCG-stimulated progesterone and estradiol production were completely restored in the presence of 10(-9) M PGF2alpha . Messenger RNA levels for the housekeeping gene beta-actin were unaltered by the above treatments . In conclusion, in the human granulosa luteal cell, PGF2alpha-R mRNA levels are inversely related to hCG-stimulated steroidogenesis (which was biphasic in nature) . Moreover, in the presence of hCG, PGF2alpha downregulates its receptor mRNA, thus providing a potential form of negative feedback on its own actions, which may be important in rescuing the corpus luteum from PGF2alpha-mediated luteolysis should pregnancy occur.

Free Radic Biol Med, 1998 Sep, 25(4-5), 621 - 8
Specific S-nitrosothiol (thionitrite) quantification as solution nitrite after vanadium(III) reduction and ozone-chemiluminescent detection; Ewing JF et al.; Increasing evidence suggests that S-nitrosothiols (thionitrites) might represent naturally occurring nitric oxide surrogates and function as intermediates in nitrogen monoxide metabolism . A facile, sensitive, and selective micromethod has been developed and validated for quantification of S-nitrosothiols as their mercury-displaceable nitrogen monoxide content . In this method, brief (5-min), room-temperature pretreatment of S-nitrosothiol with a molar excess of aqueous mercuric chloride was used to liberate into solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to its stable solution end-product, nitrite . Solution nitrite was reduced back to nitric oxide with vanadium(III), and the nitric oxide was detected by gas-phase chemiluminescence after reaction with ozone in a commercial nitric oxide analyzer . A linear relationship was observed between S-nitrosothiol-bound nitrogen monoxide and ozone-chemiluminescent detector response over a wide range (16.3-3500 pmol) of nitric oxide, as generated by reaction of vanadium(III) with either nitrite standard or mercury-treated S-nitrosothiol . Assay response was quantitatively identical for equivalent amounts of nitrite and S-nitrosothiol-bound nitrogen monoxide . The method displayed 96% selectivity for nitrite vs . nitrate and negligible (<2%) interference by nitrosated compounds bearing nitrogen monoxide moieties bound to either nitrogen or carbon . The lower limits of quantitative sensitivity and qualitative detection were below 50 and 20 pmol S-nitrosothiol-bound nitrogen monoxide-equivalents, respectively . The intraday and interday coefficients of variation did not exceed 8% . This technique has been applied to quantify structurally diverse natural and synthetic S-nitrosothiols with quantitative recovery from complex biological samples such as culture media and plasma at levels of nitrogen monoxide-equivalents undetectable by the popular Saville colorimetric method.

J Bone Miner Res, 1998 Sep, 13(9), 1370 - 7
Reduced expression of interleukin-11 in bone marrow stromal cells of senescence-accelerated mice (SAMP6): relationship to osteopenia with enhanced adipogenesis; Kodama Y et al.; Aging is associated with an increase in bone marrow adipose tissue and a reduction in bone turnover . The P6 strain of senescence-accelerated mice (SAM) exhibit an early decrease in bone mass with a reduction in bone remodeling . In the bone marrow, suppressed osteoblastogenesis and osteoclastogenesis with enhanced adipogenesis are observed . The present study was undertaken to clarify the mechanism of age-related changes in bone turnover using bone marrow cells from SAMP6 mice . Because interleukin (IL)-11 has been shown to potently inhibit adipogenesis and to stimulate osteoclast formation, the effect of IL-11 on the differentiation of bone marrow cells was examined . The impaired formation of both osteoblasts and osteoclasts was restored and the enhanced formation of adipocytes was suppressed by the addition of 10 pM recombinant human IL-11 . Other cytokines that activate gp130 as a common signal transducer, IL-6 and leukemia inhibitory factor, did not have such effects . Sequence analysis of the entire coding region of IL-11 cDNA obtained from SAMP6 stromal cells revealed no mutations . Constitutively secreted IL-11 protein into culture media, and its mRNA expression stimulated by transforming growth factor beta were reduced in stromal cells from SAMP6 compared with those in control mice . These results demonstrate that the expression of IL-11 is reduced in bone marrow cells of SAMP6 and suggest that the reduction in IL-11 actions is involved in the impairment of both osteoblastogenesis and osteoclastogenesis in these mice . There is a possibility that alterations in IL-11 actions may be associated with the age-related impairment in bone metabolism.

J Surg Res, 1998 Sep, 79(1), 13 - 9
In vitro influences between pancreatic adenocarcinoma cells and pancreatic islets; Wang F et al.; BACKGROUND: Interactions have been found between exocrine pancreatic adenocarcinoma and islets of Langerhans . Growth of pancreatic adenocarcinoma cells can be regulated by islet hormones such as insulin and somatostatin . Conversely, dysfunction of endocrine pancreas frequently accompanies the exocrine malignancy . The mechanisms underlying these interactions have not been defined . MATERIALS AND METHODS: Human pancreatic adenocarcinoma cells (HPAF cells) were cocultured with isolated rat pancreatic islets in two-compartment wells . HPAF cells and islets cultured in separate wells served as controls . In separate experiments, HPAF cells were incubated with two concentrations of exogenous insulin, including one reflecting the levels of insulin secretion seen in the coculture experiments . RESULTS: Proliferation of HPAF cells was increased by about 50% following a 2- or 5-day incubation with pancreatic islets (P < 0.05) . Coculture of HPAF cells and pancreatic islets was associated with a greater reduction in glucose concentrations (P < 0 . 01) and an increase in lactate accumulation (P < 0.05) in the culture media . Insulin concentrations in the media were significantly decreased during the first 2-3 days of the coculture incubation (P < 0.05) . In contrast, insulin secretion from control islets was not significantly decreased until the fifth day of the experiment . The growth of HPAF cells was stimulated by both concentrations of exogenous insulin (P < 0.05) . The insulin-stimulated HPAF cells also showed an enhanced glucose consumption and lactate production (P < 0.05) . CONCLUSIONS: Pancreatic islets regulate both growth and glucose metabolism of adjacent exocrine cancer cells . beta-cell-derived insulin may be one of the factors inducing these effects . Insulin release from islet beta-cells is compromised in the presence of exocrine cancer cells .

Circ Res, 1998 Sep 7, 83(5), 508 - 15
Lysophosphatidylcholine enhances cytokine-induced interferon gamma expression in human T lymphocytes; Nishi E et al.; Accumulation of substantial numbers of activated T lymphocytes, as well as monocyte/macrophages, in focal areas of arterial intima appears to be a hallmark of atherogenesis . Our previous report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherogenic lipoproteins and atherosclerotic lesions, can upregulate the expression of heparin-binding epidermal growth factor-like growth factor and the interleukin (IL)-2 receptor in cultured human peripheral T lymphocytes . In this study, we show that lyso-PC can also enhance interferon gamma (IFN-gamma) secretion and gene expression in human T lymphocytes . Lyso-PC-induced upregulation of IFN-gamma depended on the presence of IL-2, IL-12, or phytohemagglutinin in culture media and was similarly observed in both CD4+ and CD8+ subsets . Actinomycin D chase by Northern blotting showed that lyso-PC significantly prolonged IFN-gamma mRNA half-lives in human T cells . Transient transfection of IFN-gamma promoter-reporter gene construct in the human T-cell line Jurkat cells demonstrated that lyso-PC stimulated the transcription of IFN-gamma promoter-driven luciferase gene . Analyses of serial deletion mutations of IFN-gamma promoter revealed that the lyso-PC-responsive element is located between base pairs - 102 and -78 of the transcription initiation site of the IFN-gamma gene . Enhanced expression of IFN-gamma in T lymphocytes by lyso-PC may play a crucial role in atherogenesis.

Cell Immunol, 1998 Aug 1, 187(2), 151 - 7
The glycosylphosphatidylinositol-anchored form and the transmembrane form of CD58 are released from the cell surface upon antibody binding; Itzhaky D et al.; The adhesion molecule CD58 is expressed on the cell surface in both a transmembrane form and a glycosylphosphatidylinositol (GPI)-anchored form . Here we report that CD58 is released from JY cells following cross-linking by immobilized anti-CD58 monoclonal antibodies . Antibodies to other cell surface proteins, as well as PMA and LPS, did not trigger CD58 release . The release resulted from membrane cleavage, since biotin-labeled CD58 was released from biotinylated cells, and down-modulation of CD58 surface expression accompanied accumulation of soluble CD58 IN culture media . We have previously reported the isolation of JY variant cells, which lack expression of GPI anchored proteins and thus express only the transmembrane form of CD58 . Here we show that these variant cells release CD58 upon crosslinking, indicating that the transmembrane isoform is released, probably by proteolysis . Antibodies directed to the cytoplasmic domain of CD58, in contrast to antibodies against an extracellular epitope of CD58, did not react with released CD58, supporting a membrane cleavage mechanism . It is also shown that CD58, released from {3H}ethanolamine-labeled JY cells, contained ethanolamine . This result demonstrated that the GPI-anchored CD58 can be released in parallel to the transmembrane isoform and that this release does not result from proteolytic cleavage, since cleavage by a protease would have removed the ethanolamine . The present data suggest that the two isoforms of CD58 are released upon antibody binding and that their release is mediated by distinct mechanisms.

Nitric Oxide, 1998, 2(3), 155 - 64
Certain S-substituted isothioureas not only inhibit NO synthase catalytic activity but also decrease translation and stability of inducible NO synthase protein; Wei LH et al.; In an attempt to identify potent inhibitors of inducible (type II) NO synthase (iNOS) for use in cell culture systems, we found that two S-substituted isothioureas were very potent in cell culture but one such compound also interfered with the induction of NO synthase . S-Ethylisothiourea (EITU) and S-aminoethylisothiourea (AEITU) were found to be much more potent than NG-methylarginine, NG-nitroarginine methy lester, or aminoguanidine as inhibitors of NO production by cultured RAW 264.7 cell macrophages activated by lipopolysaccharide (LPS) . The approximate EC50 values as inhibitors of NO production, assessed by 24-h accumulation in cell culture media, were 10 microM (EITU), 30 microM (AEITU), 300 microM (NG-methylarginine), and 1000 microM (aminoguanidine) . EITU was found to inhibit NO production by activated macrophages without interfering with the induction of iNOS . More specifically, EITU failed to influence transcription of iNOS mRNA (Northern blot analysis), translation of iNOS protein (pulse experiments), or degradation of translated iNOS protein (pulse-chase experiments) . In contrast, however, AEITU interfered markedly with the induction of iNOS by mechanisms attributed to inhibition of translation of iNOS mRNA into functional protein as well as acceleration of degradation of already translated iNOS protein . These observations indicate that AEITU should not be used in cell culture experiments where the intent is solely to assess the consequences of inhibition of iNOS catalytic activity.

Am J Respir Cell Mol Biol, 1998 Sep, 19(3), 498 - 506
Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts; Chambers RC et al.; Chronic inhalation of cadmium at the workplace or in cigarette smoke is associated with emphysema, a disease characterized by extensive disruption of lung connective tissue . We have previously shown that cadmium, at noncytotoxic doses, inhibits fibroblast procollagen production in vitro, with maximal inhibitory effects of 69 +/- 6% (P < 0.01) at 30 &microM cadmium chloride (CdCl2) . In this paper we show that at similar doses, cadmium also inhibits proteoglycan synthesis, with values reduced by between 36 +/- 4% (P < 0.01) and 42 +/- 6% (P < 0.01) for proteoglycans secreted into the culture media and associated with the cell layer, respectively . The greatest inhibition was obtained for the major matrix-associated proteoglycans, versican, decorin, and the large heparan sulfate proteoglycans, with synthesis values reduced by between 60 and 70% . Biglycan and other heparan sulfate proteoglycans were also affected, with synthesis values reduced by between 25 and 45% . In contrast, total protein synthesis was unaffected . Furthermore, effects of cadmium at the protein level were mirrored by reduction in messenger RNA levels for alpha1(I) procollagen, versican, and decorin . These data support the hypothesis that cadmium may play an important role in the pathogenesis of emphysema associated with chronic inhalation of cadmium fumes by inhibiting the production of connective tissue proteins.

Mutat Res, 1998 Aug 3, 404(1-2), 155 - 65
Important variables that influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes-a biomarker for DNA damage in human populations; Fenech M; The cytokinesis-block micronucleus (CBMN) assay has been adopted by numerous laboratories as a means for rapidly assessing base-line chromosome damage (breakage and loss) in human populations . However, the appropriate implementation of this assay requires a thorough understanding of both experimental variables and biological factors that can have impact on micronucleus (MN) frequency . The paper describes, with the help of experimental data the from the author's laboratory as well as other data, the impact of these variables . With regards to experimental variables, the scoring of micronuclei on slides by different technicians has been identified as an important factor; however, the use of different culture media, namely RPMI 1640 and McCoy's medium, did not have a significant effect on base-line frequencies . The paper also describes results showing that the MN index in cytokinesis-blocked cells, measured once every three months over a 12-month period for 53 healthy subjects, remains constant and the data measured on these occasions were significantly and positively correlated (R=0.477 to 0.684, P<0 . 0001) with each other thus indicating the reliability and intra-individual variability of the assay over time . Inter-individual variation for males and female subjects has been estimated for each decade of age between 20 and 80 years; the difference between the 25th and 75th percentile of MN frequency varied between 1.4 fold and 2.3 fold and the minimum and maximum values for MN frequency varied by a factor of 4.7 and 12.5 depending on the age group . Age and gender are the most important demographic variables impacting on the MN index with MN frequencies in females being greater than those in males by a factor of 1.2 to 1.6 depending on the age group . For both sexes, MN frequency was significantly and positively correlated with age (R=0.62 in males and R=0.65 in females) and the slope of the regression line in males was 0.314 (P<0.0001) and in females it was 0.517 (P<0.0001) . The main dietary factors influencing the MN index in subjects who are not folate deficient are plasma B12 (R=-0.315, P=0.0127) and plasma homocysteine (R=0.415, P=0.0086) . In addition, it was proposed that the MN index is likely to be influenced by the propensity of an individual's cells to undergo apoptosis when damaged so that one might expect the MN frequency to be negatively correlated with apoptotic rate although this has yet to be tested . The above indicates the importance of maintaining an international network of scientists working with the CBMN assay to ensure appropriate quality control and for the development of standard experimental and documentation protocols . The human micronucleus (HUMN) project launched in 1997 is briefly described and proposed as the vehicle for these activities .

Virus Res, 1998 Jun, 55(2), 157 - 65
Secretion and purification of HCV E1 protein forms as glutathione-S-transferase fusion in the baculovirus insect cell system; Ciccaglione AR et al.; We have expressed the E1 protein of Hepatitis C Virus (HCV) in a new recombinant form by using a baculovirus transfer vector directing the expression of proteins fused to the carboxy-terminus of glutathione-S-transferase (GST) . The E1 domain was expressed varying at its carboxy terminus in order to retain (GST-E1) or delete (GST-E1b) the C-terminal hydrophobic region that may be involved in membrane association . Following infection with the recombinant virus, GST-E1b was efficiently secreted into the culture media and could be purified in a single step with the minimum of denaturation by glutathione affinity chromatography . The purified product was specifically immunoprecipitated by HCV positive human sera suggesting the maintenance of an immuno-relevant tertiary structure despite removal of the hydrophobic anchor . By contrast, cells infected with a recombinant baculovirus expressing GST-E1 gave a fusion protein with an appropriate molecular weight but also a series of polypeptides of lower molecular weight consistent with cleavage at the C-terminus of E1 . GST-E1 was not secreted into the medium and was associated predominantly with the membrane fraction following cell disruption; the lower molecular weight forms were soluble and secreted.

Am J Physiol, 1998 Sep, 275(3 Pt 1), G556 - 63
Induction of endothelin-1 synthesis by IL-2 and its modulation of rat intestinal epithelial cell growth; Shigematsu T et al.; Endothelin (ET), a vasoconstrictive peptide, is known to have a variety of biological actions . Although ET is released by vascular endothelial cells, other cell populations also have been reported to synthesize and release ET . In this study, we examined whether ET is synthesized by intestinal epithelial cells and whether it affects induction of epithelial cell proliferation by interleukin-2 (IL-2) . Subconfluent monolayers of intestinal epithelial cells (IEC-6 and IEC-18) were maintained in serum-free medium before addition of rat IL-2 . Both IEC-6 and IEC-18 cells released ET-1 into the medium under unstimulated conditions, as determined by a sandwich ELISA . IL-2 significantly enhanced ET-1 release in a time-dependent manner . ET-3 was not detectable in the culture media of either cell line . Expression of ET-1 and ET-3 mRNA in epithelial cells was assessed by competitive PCR . Both cell lines were shown to express ET-1 mRNA, but no ET-3 mRNA was detected . IL-2 treatment enhanced ET-1 mRNA expression by both IEC-6 and IEC-18 cells . Both cell lines also expressed mRNA for ETA and ETB receptor subtypes . When cell proliferation was assessed, exogenous ET-1 induced a slight proliferative response in both types of cells that was consistent and significant at low ET-1 concentrations; cell growth was inhibited at a higher concentration (10(-7) M) . IL-2 produced a significant proliferative response in both cell lines . However, the addition of ET-1 (10(-7) M) to culture media attenuated the IL-2-induced increase in cell proliferation . ETA-receptor antagonists significantly enhanced cellular proliferation, suggesting involvement of the ETA receptor in modulation of IL-2-induced intestinal epithelial cell growth.

Endocrinology, 1998 Sep, 139(9), 3837 - 42
Growth hormone and its receptor are expressed in human thymic cells; de Mello-Coelho V et al.; GH has been shown to modulate various functions of the thymus . We now demonstrate the production of human GH (hGH) by human thymic cells, and the expression of GH receptors in thymic epithelial cells (TEC) and in thymocytes at different stages of differentiation . The presence of hGH messenger RNA was shown by RT-PCR in both human thymocytes and in primary cultures of TEC . Moreover, immunoreactive hGH material was detected in culture media of thymocytes and TEC with the use of a sensitive immunoradiometric assay . GH receptor gene expression was shown in TEC in primary cultures and in fetal and postnatal TEC lines as well as in thymocytes . By immunocytochemistry, the presence of GH receptors in the various TEC preparations was confirmed . In cytofluorometric studies with the use of a biotinylated anti-GH receptor monoclonal antibody, we could show that GH receptors are predominantly expressed by immature thymocytes: over 90% of CD3- CD4- CD8- CD19- CD34+ CD2- cells (a phenotype characterizing the most immature T cell progenitors in the thymus) were GH receptor positive . Our results provide a molecular basis for an autocrine/paracrine mode of action of GH in the human thymus.

Arch Dermatol, 1998 Aug, 134(8), 955 - 60
Physician-diagnosed erythema migrans and erythema migrans-like rashes following Lone Star tick bites; Masters E et al.; OBJECTIVE: To differentiate cases of physician-diagnosed erythema migrans and erythema migrans-like rashes associated with Lone Star tick (Amblyomma americanum) bites . DESIGN: Retrospective case series . SETTING: Private primary care clinic in rural Missouri . PATIENTS: Seventeen patients with physician-diagnosed erythema migrans following a definite Lone Star tick bite at the rash site . INTERVENTIONS: A biopsy was performed on all rash sites . All patients were treated with oral antibiotics . MAIN OUTCOME MEASURES: Rash appearance, size, body location, multiple lesions, incubation times, associated symptoms, seasonal occurrence, histopathological features, tick stage and sex, patient age and sex, treatment response, growth in BSK II culture media, and serologic evaluation . RESULTS: Rashes associated with Lone Star ticks were similar to erythema migrans vectored by other Ixodes ticks . Differences were noted in Lyme disease serology results, especially flagellin-based enzyme immunoassays, and failure to yield spirochetes in BSK II cultures . Lyme serology results were often negative, but were also frequently inconsistent with results of controls without Lyme disease . CONCLUSIONS: Lone Star ticks are associated with rashes that are similar, if not identical, to erythema migrans associated with borrelial infection . The recent isolation and cultivation of Borrelia burgdorferi from ticks (including 1 Lone Star tick) from the farm of a patient included in this report has raised the possibility that Lone Star ticks are "bridge vectors" for human borrelial infection . Although further investigation is needed, these rashes may be secondary to spirochetal infection.

Cancer Res, 1998 Aug 15, 58(16), 3627 - 32
Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme; Potter PM et al.; Enzyme activation of prodrugs to improve the therapeutic index of specific anticancer agents is an attractive alternative to current chemotherapy regimens . This study addresses the potential for activating irinotecan (CPT-11) with recombinant carboxylesterases (CEs) . CEs are a ubiquitous class of enzymes thought to be involved in the detoxification of xenobiotics . Their primary amino acid sequence indicates that these proteins should be localized to the endoplasmic reticulum . By PCR-mediated mutagenesis of a rabbit liver and a human alveolar macrophage CE cDNA, expression in Cos7 cells, and subsequent immunohistochemical localization, we have determined that an 18-amino acid NH2-terminal hydrophobic signal peptide is responsible for the localization of these proteins to the endoplasmic reticulum . By similar approaches, we have demonstrated that the COOH-terminal amino acids HIEL prevent secretion of the proteins from the cell . Enzymatic activity was lost by removing the NH2-terminal domain; however, active enzyme could be detected in the culture media of cells expressing the COOH-terminally truncated proteins . Secretion of CEs lacking the six COOH-terminal amino acids could be prevented with brefeldin A, confirming that these truncated enzymes were processed and released from cells by endoplasmic reticulum-mediated exocytosis . Double-truncation mutant enzymes lacking both NH2- and COOH-terminal sequences demonstrated immunostaining patterns similar to those of the NH2-terminally truncated proteins and also lacked CE activity . In all cases, metabolism of the classic esterase substrate o-nitrophenyl acetate predicted the sensitivity of cells expressing the rabbit CE to the anticancer agent CPT-11 . In addition, the secreted enzyme sensitized Cos7 cells to this drug, indicating that protein association with a lipid bilayer is not required for substrate metabolism.

Biomaterials, 1998 Jul, 19(13), 1189 - 95
Synthesis of sulfonylurea conjugated copolymer via PEO spacer and its in vitro short-term bioactivity in insulin secretion from islets of Langerhans; Hwang JS et al.; In order to reduce the number of immunoprotected islets required in xeno- or allogenic transplants for reversing diabetes, analogues of glyburide (a sulfonylurea), an extremely hydrophobic insulin secretagogue, were synthesized and used in an attempt to produce water soluble sulfonylurea (SU) grafted polymers . After synthesizing various polymers containing glyburide analogues, a poly(N-vinyl-2-pyrrolidone-co-sulfonylurea succinyl PEO (Mw = 3000) acrylate) was found to be soluble in a cell culture medium at pH 7.4 . However, solubility was only obtained by decreasing solution pH from 11 to 7.4 . When the copolymer was added to the islet cell culture media at a concentration of 5 microg ml(-1) (based on the theoretical SU content of the copolymer), insulin secretion was enhanced by about 30% at low glucose concentrations of 50 and 100 mg dl(-1) compared to the control . This is equivalent to 40-60% bioactivity of glyburide . The polymer's effect on insulin secretion at a higher glucose concentration of 200 mg dl(-1) was not significant . Considering the previous results where a similar but insoluble polymer without a PEO spacer was used and the polymer showed SU bioactivity only at a glucose concentration of 50 mg dl(-1), the observations from this study indicates that the solubility of SU-grafted polymers may affect the binding of SU groups to SU receptors on the pancreatic beta-cells, resulting in improved pharmacodynamic effect of SU.

Leuk Lymphoma, 1998 Sep, 31(1-2), 61 - 9
Role of umbilical vein endothelial cells in hematopoiesis; Yamaguchi H et al.; Effective hematopoiesis is usually induced by interactions between hematopoietic progenitor cells (HPC) and stromal cells . In cord blood (CB), umbilical vein endothelial cells (HUVEC) can support HPC as a stromal microenvironment . EC activated mainly by IL-1 and TNFalpha produce a variety of cytokines and growth factors such as IL-1, IL-4, IL-6, GM-CSF and G-CSF . Since HPC express c-kit on their surface, the SCF produced by HUVEC plays an important role in the hematopoiesis of CB . We examined the expression of cytokines and growth factors on HUVEC by PCR . Resting HUVEC expressed high level of SCF, and low levels of IL-6, IL-7, and IL-8 . Thus, a variety of cytokines and growth factors are produced by EC, and this cytokine network is thought to play an important role in regulating hematopoiesis . Activated EC can also express various adhesion molecules including E-selectin, VCAM-1 and ICAM-1, and facilitate the adhesion of hematopoietic cells to the endothelium . Furthermore, the interaction of CB cells with HUVEC has recently been shown in vitro . We previously showed that the culture media of HUVEC induced high numbers of colony formation . Suitable cytokine productions are thus provided to HPC by the interaction of HUVEC and cord MNC . On the basis of these findings, several mechanisms to support hematopoiesis in CB can be considered . Specific growth factors produced by EC bind to HPC to induce proliferation . While cell-cell interactions involve adhesion of HPC to HUVEC via adhesion molecules, and the adhesion of HPC to EC will facilitate interaction with cytokines and growth factors . Thus HPC in CB proliferate and are maintained by growth factors, and adhesion molecules produced by HUVEC, and HPC themselves.

Biol Reprod, 1998 Sep, 59(3), 632 - 42
Characterization of insulin-like growth factor-binding proteins in the uterus and conceptus during early conceptus elongation in cattle; Keller ML et al.; As a first step in determining the role that insulin-like growth factor (IGF)-binding proteins (BPs) may have in regulating initial stages of conceptus elongation in cattle, the type and relative abundance of IGFBPs in serum, uterine tissues, and uterine fluid from pregnant and noninseminated cows on Days 13 and 15 postestrus and in Day 15 conceptuses was evaluated . Uterine and serum samples contained IGFBPs 2, 3, 4, and 5 as determined by immunoprecipitation followed by Western ligand blots of precipitates . Compared with those in uterine and serum samples, IGFBPs in conceptuses and conceptus-conditioned culture media were only faintly detectable . The percentage of the total IGF-I binding activity attributed to IGFBP-3 was greater (p < 0.05) in myometrium, serum, and uterine fluid (> 50%) than in inter- (40%) and intracaruncular (37%) endometrium . Percentage of total binding attributed to IGFBP-2 was greater (p < 0.05) in endometrium and serum ( approximately 30%) than in myometrium (16%) and uterine fluid (9%) . Binding activity of certain IGFBPs varied due to day of the estrous cycle or due to pregnancy status . Concentrations of IGF-I in serum were greater (p < 0.05) in nonpregnant (52 +/- 2 ng/ml) than in pregnant (40 +/- 4 ng/ml) cows . Concentration of IGF-I in uterine fluid did not differ due to pregnancy status or stage of cycle (4.4 pg IGF-I/ microg uterine protein) . Northern blots revealed mRNAs for IGFBPs 1, 2, 3, 4, and 5 in uterine tissues but not in conceptuses . In situ hybridization indicated that IGFBP-1 mRNA was primarily localized in luminal epithelium of endometrium; IGFBP-2 mRNA was in luminal epithelium and dense stromal cells adjacent to endometrial epithelium; and IGFBP-3 mRNA was in vascular endothelial cells and was more prevalent in myometrium than in endometrium . Tissue specificity and changes in abundance of IGFBPs in the uterus during early conceptus elongation indicate the potential importance of IGFBP regulation of uterine IGFs during this time period.

J Lipid Res, 1998 Aug, 39(8), 1629 - 40
Inhibition of N-linked glycosylation results in retention of intracellular apo{a} in hepatoma cells, although nonglycosylated and immature forms of apolipoprotein{a} are competent to associate with apolipoprotein B-100 in vitro; Bonen DK et al.; Apolipoprotein{a} (apo{a}) is a highly polymorphic glycoprotein that forms a covalent complex with apolipoprotein B-100 (apoB-100), producing a lipoprotein species referred to as lipoprotein{a} (Lp{a}) . We have studied the effects of alterations in glycosylation of apo{a} on its intracellular processing and secretion as well as its ability to associate with low density lipoprotein (LDL) apoB-100 . HepG2 cells transfected with a 6 kringle IV (6 K-IV) apo{a} minigene were treated with tunicamycin, an inhibitor of N-linked glycosylation, which eliminated apo{a}-B-100 complexes from the media . Tunicamycin treatment also reduced secretion of the 6 K-IV apo{a} protein from transfected McA-RH7777 cells by approximately 50%, but completely eliminated secretion of apo{a} species containing 9 and 17 K-IV repeats . Mixing experiments, performed with radiolabeled media (+/-tunicamycin) from transfected McA-RH7777 cells, demonstrated no alteration in the extent of association of apo{a} with human LDL . Similar mixing experiments using culture media from glycosylation-defective mutant chinese hamster ovary (CHO) cells transfected with the same apo{a} minigene showed identical results . Apo{a} secretion was demonstrated in all mutant cell lines in the absence of either N- or O-linked (or both) glycosylation . The mechanisms underlying the reduced secretion of apo{a} from transfected hepatoma cells were examined by pulse-chase radiolabeling and apo{a} immunoprecipitation . Tunicamycin treatment altered the efficiency of precursor apo{a} processing from the ER by increasing its ER retention time . The increased accumulation of precursor apo{a} in the ER was associated with alterations in the kinetics of association with two resident endoplasmic reticulum (ER) chaperone proteins, calnexin and BiP . These findings suggest that the glycosylation state and size of apo{a} appear to play a role in regulating its efficient exit from the endoplasmic reticulum . However, neither N- nor O-linked glycosylation of apo{a} exerts a major regulatory role in its covalent association with apoB-100.

J Rheumatol, 1998 Aug, 25(8), 1595 - 601
Nitric oxide downregulates interleukin 1beta (IL-1beta) stimulated IL-6, IL-8, and prostaglandin E2 production by human chondrocytes; Henrotin YE et al.; OBJECTIVE: To investigate the effects of endogenously produced nitric oxide (NO) on interleukin 6 (IL-6), IL-8, prostaglandin E2 (PGE2), and proteoglycan production by human chondrocytes . METHODS: Human articular chondrocytes were isolated from their extracellular matrix by triple successive enzymatic digestion of the cartilage and cultured 48 h in a well defined culture medium . IL-6 and IL-8 were directly assayed into culture media by specific enzyme amplified sensitivity immunoassays . Proteoglycans and PGE2 were quantified by specific radioimmunoassays . Cell culture media were assayed for NO2 using a spectrophotometric assay based upon the Griess reaction . RESULTS: Unstimulated chondrocytes produced low levels of NO, IL-6, IL-8, and PGE2 . Production was significantly stimulated by IL-1beta and lipopolysaccharide (LPS) . As well, proteoglycan synthesis was profoundly inhibited by IL-1beta and LPS . Inhibition of NO synthesis with the competitive inhibitor NG-monomethyl-L-arginine (L-NMMA) led to enhancement of IL-6, IL-8, and PGE2 production stimulated by either IL-1beta alone or in combination with LPS, whereas the inhibition of proteoglycan production by IL-1beta was not modified by L-NMMA . CONCLUSION: LPS and IL-1beta stimulated IL-6, IL-8, and PGE2 production are downregulated by endogenously produced NO, which could limit the inflammatory reaction occurring in arthritis.

Pigment Cell Res, 1998 Aug, 11(4), 189 - 97
Anterior/posterior influences on neural crest-derived pigment cell differentiation; Thibaudeau G et al.; The neural crest of vertebrate embryos has been used to elucidate steps involved in early embryonic cellular processes such as differentiation and migration . Neural crest cells form a ridge along the dorsal midline and subsequently they migrate throughout the embryo and differentiate into a wide variety of cell types . Intrinsic factors and environmental cues distributed along the neural tube, along the migratory pathways, and/or at the location of arrest influence the fate of neural crest cells . Although premigratory cells of the cranial and trunk neural crest exhibit differences in their differentiation potentials, premigratory trunk neural crest cells are generally assumed to have equivalent developmental potentials . Axolotl neural crest cells from different regions of origin, different stages of development, and challenged with different culture media have been analyzed for differentiation preferences pertaining to the pigment cell lineages . We report region-dependent differentiation of chromatophores from trunk neural crest at two developmental stages . Also, dosage with guanosine produces region-specific influences on the production of xanthophores from wild-type embryos . Our results support the hypothesis that spatial and temporal differences among premigratory trunk neural crest cells found along the anteroposterior axis influence developmental potentials and diminish the equivalency of axolotl neural crest cells.

Exp Clin Endocrinol Diabetes, 1998, 106(3), 203 - 10
Molecular cloning of pit-1 cDNA from porcine anterior pituitary and its involvement in pituitary stimulation by growth hormone-releasing factor; Chung HO et al.; Pit-1/GHF-1 (hereafter Pit-1) is a pituitary-specific transcription factor that participates in growth hormone (GH), prolactin and thyroid-stimulating hormone gene expression and in proliferation of the cells that produce these hormones . In this study, we determined the nucleotide sequence of porcine Pit-1 cDNA, which shows high homology (95%) with the known mammalian Pit-1s . Some differences in amino acid sequence were found in the amino terminal region, but the POU-specific and POU-homeo domains were well conserved . Porcine anterior pituitary cells in primary culture were treated for 24 h with GH-releasing factor (GRF), forskolin and phorbol ester (TPA) . These agents stimulated progressive secretion of GH into the culture media . The levels of Pit-1, GH, cJun and cFos mRNAs were examined using the reverse transcription-polymerase chain reaction . Stimulation with GRF for 2 h increased the Pit-1 mRNA level 3-fold . This response was transient, as the level returned to the control level after 6 h . Forskolin and TPA evoked similar increases, but their effects appeared after 30 min and reached their maxima after 2 h . In contrast, GRF and forskolin, but not TPA, increased the GH mRNA level 2-fold after 24 h . The cJun mRNA level showed no significant change in response to these agents over 24 h and GRF and TPA increased the cFos mRNA level 1.4 and 2.3-fold, respectively, after 30 min . These results suggest that increasing Pit-1 mRNA level in response to GRF plays a role in the early event of the GH gene expression or with the assistance of other transcription factors that modulate GH gene . Furthermore, the GRF-induced increase in cFos mRNA level by GRF and TPA did not appear to be participated straightforward in the GH gene expression, suggesting that cFos alone is insufficient to the activation.

Cardiovasc Res, 1998 May, 38(2), 516 - 21
Release of chemoattractants for human monocytes from endothelial cells by interaction with neutrophils; Schratzberger P et al.; OBJECTIVE: The release of monocyte chemoattractant protein-1 (MCP-1) in the vessel wall may lead to accumulation of monocytes in the subendothelial space . The role of neutrophils (PMNL) in the initiation of this process is unknown . We tested whether PMNL are able to induce the production and release of MCP-1 in endothelial cells . METHODS: PMNL were allowed to interact with human umbilical vein endothelial cell (HUVEC) monolayers in culture . Culture media were collected and assessed for chemotactic activity on mononuclear leukocytes (MNC) or purified monocytes in a modified Boyden chamber assay . Additionally, MCP-1 levels in supernatants were quantified by ELISA . RESULTS: Media from unstimulated HUVEC culture supernatants induced a slight increase (1.2-fold) of MNC and purified monocyte chemotaxis, which was significantly augmented by addition of PMNL for 1 h (1.4-fold; P < 0.05) . The increase in chemotaxis was time- and dose-dependent and could be blocked by an anti-MCP-1 monoclonal antibody . Media obtained after coculture of PMNL and HUVEC for 1-5 h contained increased amounts of MCP-1 as measured by ELISA; addition of cycloheximide abolished this response . CONCLUSIONS: Interaction of PMNL with endothelium induces the release of functionally active MCP-1 suggesting that in the vascular wall, PMNL may play a role in the recruitment of MNC.

J Food Prot, 1998 Jun, 61(6), 728 - 30
Gamma irradiation and ozone treatment for inactivation of Escherichia coli O157:H7 in culture media; Byun MW et al.; A study was conducted to investigate the reduction and elimination of Escherichia coli O157:H7 by the effects of gamma irradiation and ozone treatment . Log phase cells were found to be more sensitive to gamma irradiation than stationary phase cells . E . coli O157:H7 was found to be considerably more resistant to irradiation at -18 degrees C than at 20 degrees C . The D values for this organism for treatment with ozone in tryptic soy agar were higher than those for treatment with ozone in phosphate buffer . Gamma irradiation at a dose of 1.5 kGy or ozone treatment at a concentration of 3 to 18 ppm for 20 to 50 min was required to assure the elimination of E . coli O157:H7.

Arch Dermatol Res, 1998 Jun, 290(6), 342 - 9
Variations in melanin formation by cultured melanocytes from different skin types; Smit NP et al.; In many laboratories, culturing skin melanocytes has become a routine research activity . However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells . To shed more light on this issue, we examined the influence of different culture media on pigment production . We showed that there were notable passage-to-passage variations in the synthesis of melanin . This was particularly true for phaeomelanin . It is therefore advisable to analyse the melanin in the cells before the start of experiments . In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy . Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures . In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes . With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested . This stimulation was particularly prominent in skin type I melanocytes . Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.

Endocrine, 1998 Apr, 8(2), 185 - 91
Regulation of human luteal steroidogenesis in vitro by nitric oxide; Vega M et al.; To evaluate the effect of nitric oxide (NO.) in human corpus luteum (CL) function, we investigated the expression and the presence of NO . synthase (NOS) in the human CL and the action of NO . on the in vitro luteal steroid production . The expression of endothelial NOS (eNOS) in early, mid-, and late CL was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and the immunohistochemical study was performed in human CL histological sections by using monoclonal antibodies (MAbs) against the distinct NOS isoforms . In addition, seven human mid-CLs were enzymatically dispersed, and the cells were cultured with NO . donor compounds . Steroid production was measured in the culture media by specific radioimmunoassay . The results show that the expression of eNOS was highly detected in mid- and early CL, and to a lesser extent, in late CL . Meanwhile, the immunohistochemical study indicated that both isoenzymes of NOS were expressed in mid-human CL, eNOS being the more abundant isoform present . On the other hand, functional studies showed that NO . donors (L-arginine {L-Arg} and sodium nitroprusside) elicited an inhibitory action on steroid synthesis, preferentially on estradiol production by the luteal cell cultures (p < 0.05) . In conclusion, the NO-NOS system is present in the human CL, and it may serve as a modulator of the in vitro human luteal steroidogenesis.

Anticancer Res, 1998 Jul-Aug, 18(4A), 2397 - 403
Cyclooxygenase-2 expression in human adenocarcinoma cell line HT29 cl.19A; Battu S et al.; BACKGROUND: Overexpression of cyclooxygenase-2 (COX-2) has been implicated in carcinogenesis of human colorectal cancer which is one of the leading types of cancer in Western countries . MATERIALS AND METHODS: We analyzed the COX-2 expression and activity using RT-PCR, Western blot, immunocytochemistry, RP-HPLC and EIA analysis in 0% and 10% fetal bovine serum (FBS) cultured cells . RESULTS: HT 29 cl.19A cells exhibited a COX-2 expression called "constitutive" in the absence of FBS in culture media . This particular expression was not the result of a mutation of the HT29 cl.19A COX-2 gene promotor . CONCLUSION: In our study, the human colon adenocarcinoma cell line, HT29 cl.19A, expressed COX-2 abnormally . This expression appeared to be at the same time inducible by the action of classical exogenous inducers such as FBS or interleukin-1 beta and "constitutive" if none of these compounds were present.

J Cell Physiol, 1998 Sep, 176(3), 516 - 24
Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes; Boyan BD et al.; Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells . In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC . Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC . To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine {OEPC}), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite . PKC specific activity in the cell layer was determined as a function of time . Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC . When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased . Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC . For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells . These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity . Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2 . These effects are cell maturation- and time-dependent and metabolite-specific.

Am J Physiol, 1998 Jun, 274(6 Pt 1), G1077 - 86
Pancreatic trypsinogen I expression during cell growth and differentiation of two human colon carcinoma cells; Bernard-Perrone F et al.; Pancreatic trypsin has been found to induce tight junction or dome formation in some colon cancer cell lines (HT-29, Caco-2), and a tumor-associated trypsinogen, trypsinogen type II, has been isolated from another colon cancer cell line (COLO 205) . We have tried to determine if trypsinogen is present and how its expression varies during cell culture in HT-29 Glc+/- and Caco-2 cells, which exhibit enterocytic differentiation, and in HT-29 Glc+ cells, which never differentiate . Trypsinogen mRNA presence and expression were demonstrated in these cells by mRNA hybridization, RT-PCR, cytoimmunofluorescence, Western immunoblot analysis, and gel filtration . Trypsinogen was found to be trypsinogen type I and was mainly in zymogen form in culture media . Differentiating cells exhibited variations in trypsinogen I expression, but cells that remained undifferentiated did not . In the differentiated cells, a high and transient peak in trypsinogen I expression was observed during the first steps of differentiation.

Am J Physiol, 1998 Jun, 274(6 Pt 1), G1068 - 76
Regulation of endothelin synthesis in hepatic endothelial cells; Eakes AT et al.; Endothelin (ET) stimulates vasoconstriction and glucose production and mediator synthesis in the liver . Only hepatic endothelial cells express ET-1 mRNA, and during endotoxemia in the intact rat, a ninefold increase in hepatic ET-1 mRNA occurs within 3 h of lipopolysaccharide (LPS) infusion {A . T . Eakes, K . M . Howard, J . E.Miller, and M . S . Olson . Am . J . Physiol . 272 (Gastrointest . Liver Physiol . 35): G605-G611, 1997} . The present study defines the mechanism by which hepatic ET production is enhanced during endotoxin exposure . Culture media conditioned by exposure to endotoxin-treated Kupffer cells stimulated a twofold increase in immunoreactive ET-1 (irET-1) secretion by liver endothelial cells . Transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), LPS, and platelet-activating factor (PAF) were tested for their ability to stimulate cultured liver endothelial cells to secrete irET-1 . Although TNF-alpha, LPS, and PAF had no significant effect on ET-1 synthesis, TGF-beta increased ET-1 mRNA expression and irET-1 secretion . In coculture experiments, treating Kupffer cells with endotoxin caused a doubling of the ET-1 mRNA level in the liver endothelial cells.This increase in ET-1 mRNA was attenuated by a TGF-beta-neutralizing antibody . Hence, a paracrine signaling mechanism operates between Kupffer cells that release TGF-beta on endotoxin challenge and hepatic endothelial cells in which TGF-beta stimulates ET-1 mRNA expression and ET-1 secretion; this intercellular signaling relationship is an important component in the hepatic responses to endotoxin exposure.

Parasitology, 1998 Jul, 117 ( Pt 1), 39 - 47
Inhibition of the development of Eimeria tenella in cultured bovine kidney cells by a soluble factor produced by peripheral blood lymphocytes from immune chickens; Bumstead JM et al.; The intracellular development of Eimeria tenella sporozoites in in vitro cultured Madin-Darby Bovine Kidney (MDBK) cells was inhibited when parasite-infected MDBK cells were incubated with peripheral blood lymphocytes (PBL) from infected chickens . The inhibition mediated by PBL was quantified by {3H}uracil uptake and increased during the course of a series of oral infections of chickens with E . tenella . This was mirrored by the development of immunity in these birds, as assessed by counting the oocyst output following each re-infection . Similar levels of inhibition were observed using PBL from 3 inbred lines of chickens which differ in their relative susceptibility to infection with E . tenella, indicating that the genetic background of the host does not influence the production of this inhibitory activity . The inhibition could be transferred to freshly infected MDBK cells using supernatants prepared from parasite-infected monolayers incubated for 48 h with PBL from immune chickens . However, there was no inhibition using either supernatants from infected MDBK cells incubated with PBL from uninfected chickens, or supernatants from uninfected MDBK cells incubated with PBL from immune chickens . Experiments using Transwell plates showed that direct contact of PBL from immune birds with infected MDBK monolayers was not required to produce supernatants with inhibitory activity . Thus production of soluble inhibitory factor(s) by PBL from immune chickens can be specifically induced by soluble antigens present in the culture media of parasite-infected MDBK cells . These factors inhibit the intracellular development of sporozoites in in vitro culture.

Electrophoresis, 1998 Jun, 19(8-9), 1475 - 7
Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms; Arcelloni C et al.; Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described . Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm X 50 microm internal diameter) . Proteins were monitored at 200 nm and separated at constant voltage . Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE . Protein recovery was 85+/-14% (n = 5, mean+/-standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL . Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.

Biomaterials, 1998 Jun, 19(11-12), 999 - 1007
Effects of AISI 316L corrosion products in in vitro bone formation; Morais S et al.; Rat bone marrow cells were cultured in experimental conditions that favour the proliferation and differentiation of osteoblastic cells (i.e., 2.52 x 10(-4) mol l(-1) ascorbic acid, 10(-2) mol l(-1) beta-glycerophosphate and 10(-8) mol l(-1) dexamethasone) in the absence and in the presence of stainless-steel corrosion products, for a period of 18 days . An AISI 316L stainless-steel slurry (SS) was obtained by electrochemical means and the concentrations of the major metal ions, determined by atomic absorption spectrometry, were 8.78 x 10(-3) mol l(-1) of Fe, 4.31 x 10(-3) mol l(-1) of Cr and 2.56 x 10(-3) mol l(-1) of Ni . Bone marrow cells were exposed to 0.01, 0.1 and 1% of the SS and at the end of the incubation period, control and treated cultures were evaluated by histochemical assays for the identification of the presence of alkaline phosphatase and also calcium and phosphate deposition . Cultures were further observed by scanning electron microscopy . Levels of total and ionised calcium and phosphorus in the culture media collected from control and metal exposed cell cultures were also quantified . Histochemical staining showed that control cultures presented a strong reaction for the presence of alkaline phosphatase and exhibited formation of calcium and phosphates deposits . The presence of 0.01% SS caused no detectable biological effects in these cultures, 0.1% SS impaired osteoblastic behaviour and, 1% SS resulted in cell death . In the absence of bone cells, levels of total and ionised calcium and phosphorus in the control and metal added culture medium were similar throughout the incubation period . A significant decrease in the levels of ionised calcium and phosphorus were observed in the culture medium of control cultures and also in cultures exposed to 0.01% SS after two weeks of incubation, an event related with the formation of mineral calcium phosphate deposits in these cultures . In cultures grown in the presence of 0.1 and 1% SS corrosion products, levels of calcium and phosphorus were similar to those observed in the absence of cells . Results showed that stainless-steel corrosion products above certain concentrations may disturb the normal behaviour of osteoblast-like rat bone marrow cell cultures.

J Endocrinol, 1998 Jun, 157(3), 453 - 61
Tri-iodothyronine regulates the production of interleukin-6 and interleukin-8 in human bone marrow stromal and osteoblast-like cells; Siddiqi A et al.; Hyperthyroidism is associated with increased bone resorption but the mechanisms by which thyroid hormone (T3) affects bone cell metabolism remain unclear . Recently it has been suggested that T3 stimulates osteoclastic resorption indirectly through the release of soluble mediators from osteoblasts . The aim of the present study was to investigate whether the T3-induced increase in bone resorption could be due to the regulation of cytokine production by human osteoblasts (hOb) . The effects of T3 (1, 10, 100 nM) and IL-1 beta (100 U/ml) as the positive control were examined on cytokine protein release and mRNA levels in cultured hOb cell lines (MG63, SaOs-2), primary hOb and human bone marrow stromal (hBMS) cells . T3 increased IL-6 and IL-8 mRNA levels as well as IL-6 and IL-8 protein release into the culture media from MG63 and hBMS cells in a time- and dose-dependent manner . The maximal effect on protein release in hBMS cells occurred at 24 h with a dose of T3 10 nM (IL-6 5.5 +/- 1.1-fold above controls; IL-8 3.7 +/- 0.5-fold above controls, P < 0.05) . At the same time, mRNA levels in hBMS cells were increased 6.2 +/- 0.8-fold for IL-6 (P < 0.05) and 5.7 +/- 0.8-fold for IL-8 (P < 0.05) . Similar results were obtained in MG63 cells but no response was seen in SaOs-2 or hOb cells despite measurable basal production . Nor was there detectable regulation of IL-1 beta, IL-3, IL-11, IL-4 or granulocyte macrophage-colony stimulating factor by T3 in any cell type . In conclusion, T3 increases IL-6 and IL-8 production by MG63 and hBMS cells, suggesting that IL-6 and IL-8 may be T3-regulated genes in osteoblasts.

IEEE Trans Biomed Eng, 1998 Aug, 45(8), 1067 - 76
Finite-difference time-domain analysis of a complete transverse electromagnetic cell loaded with liquid biological media in culture dishes; Popovic M et al.; Transverse electromagnetic (TEM) cells can be used for exposing biological culture specimens to electromagnetic fields and observing possible anomalous effects . The uniformity of field exposure is critical to quantifying the biological response versus the electromagnetic dose . Standing waves and other electromagnetic field nonuniformities can cause nonuniform exposure . This paper reports the results of high-resolution three-dimensional finite-difference time-domain (FDTD) simulations of a complete TEM cell designed for operation at 837 MHz . Several different cases were studied in which the number of culture dishes, the depth of the culture liquid, and the orientation of the culture dishes were varied . Further, the effect of the culture-dish glass bottom thickness and the meniscus of the liquid medium were examined . The FDTD results show that there is a significant nonuniform field and specific absorption rate (SAR) distribution within the culture medium for each case examined . Hence, biological dose-response experiments using the TEM cells should account for the possibility of strong localized SAR peaking in the culture media to provide useful data in setting exposure standards for wireless communications.

Biochim Biophys Acta, 1998 Jun 16, 1398(2), 203 - 14
Sequence and expression analysis of bovine pigment epithelium-derived factor; Perez-Mediavilla LA et al.; PEDF, a member of the serpin superfamily of proteins related through their highly conserved folded conformation, has neurotrophic properties, including promotion of neurite-outgrowth and neuronal survival . Previously, we have purified and characterized PEDF protein from extracellular matrixes of bovine eyes . Here, we show the cDNA sequence and expression analysis of bovine PEDF . Northern analysis of RNA from bovine retinal pigment epithelium (RPE) and neural retina using a human PEDF cDNA fragment reveals expression of the PEDF gene only for RPE . Sequence analysis of a cDNA clone isolated from bovine RPE predicts a polypeptide of 416 amino acid residues that shares 88.6% and 85% amino acid identity with human and mouse PEDF, respectively . It has an N-terminal signal peptide, a consensus glycosylation site and homology with serpins including the conserved residues required for maintaining the serpin tertiary structure . Cell-free expression of the bovine PEDF cDNA by in vitro transcription and translation yields a precursor polypeptide of 45,000-Mr that immunoprecipitates with an antibody to human PEDF . Expression analysis in stably transfected baby hamster kidney cells shows that the recombinant bovine protein is secreted to the culture media as a mature 50,000-Mr protein, which induces neurite-outgrowth on retinoblastoma cells, like the naturally-occurring PEDF protein . Thus, the bovine PEDF cDNA isolated here codes for a functional soluble secreted PEDF glycoprotein.

Am J Physiol, 1998 Aug, 275(2 Pt 1), C619 - 21
Using gadolinium to identify stretch-activated channels: technical considerations; Caldwell RA et al.; Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes . Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+ insensitivity has been used to infer that processes are not dependent on SACs . The limitations of this approach are not adequately appreciated, however . Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account . Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs . Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels . Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.

Eur J Haematol, 1998 Jul, 61(1), 1 - 6
Comparison of methods for retroviral mediated transfer of glucocerebrosidase gene to CD34+ hematopoietic progenitor cells; Takiyama N et al.; Gaucher disease is an excellent candidate for gene therapy by transduction of hematopoitic stem cells . In this study, we compared methods which allow an increase in transfer of the glucocerebrosidase gene to human hematopoietic progenitor cells . Several techniques were employed, including the use of cytokines, bone marrow stroma, fibronectin, centrifugal enhancement and in vitro long-term culture . The effect of prestimulation with cytokines interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) on transduction of cord blood CD34+ cells was examined . The results suggest that 16-h prestimulation was sufficient for efficient transduction . We examined the effect of bone marrow stroma and fibronectin, both of which increased transduction efficiency up to 36% and 44%, respectively, as measured by PCR for the integrated GC-cDNA in clonogenic cells (9% without any support) . Transduction efficiency of 83% was obtained using 2-h centrifugation . Combining centrifugation and in vitro culture in long-term bone marrow culture media containing cytokines (IL-3/IL-6/SCF), CD34+ cells from cord blood and peripheral blood of 3 Gaucher patients were transduced weekly for 21 d . The results of 6 separate experiments consistently demonstrated transduction efficiency of 100% after 7-d in vitro culture . This transduction protocol combining centrifugation and in vitro long-term culture is an attractive method and can be applied to clinical trials.

EMBO J, 1998 Aug 3, 17(15), 4291 - 303
The thyroid hormone receptor functions as a ligand-operated developmental switch between proliferation and differentiation of erythroid progenitors; Bauer A et al.; The avian erythroblastosis virus (AEV) oncoprotein v-ErbA represents a mutated, oncogenic thyroid hormone receptor alpha (c-ErbA/ TRalpha) . v-ErbA cooperates with the stem cell factor-activated, endogenous receptor tyrosine kinase c-Kit to induce self-renewal and to arrest differentiation of primary avian erythroblasts, the AEV transformation target cells . In this cooperation, v-ErbA substitutes for endogenous steroid hormone receptor function required for sustained proliferation of non-transformed erythroid progenitors . In this paper, we propose a novel concept of how v-ErbA transforms erythroblasts . Using culture media strictly depleted from thyroid hormone (T3) and retinoids, the ligands for c-ErbA/TRalpha and its co-receptor RXR, we show that overexpressed, unliganded c-ErbA/ TRalpha closely resembles v-ErbA in its activity on primary erythroblasts . In cooperation with ligand-activated c-Kit, c-ErbA/ TRalpha causes steroid-independent, long-term proliferation and tightly blocks differentiation . Activation of c-ErbA/ TRalpha by physiological T3 levels causes the loss of self-renewal capacity and induces synchronous, terminal differentiation under otherwise identical conditions . This T3-induced switch in erythroid progenitor development is correlated with a decrease of c-ErbA-associated histone deacetylase activity . Our results suggest that the crucial role of the mutations activating v-erbA as an oncogene is to 'freeze' c-ErbA/ TRalpha in its non-liganded, repressive conformation and to facilitate its overexpression.

Genome Res, 1998 Jul, 8(7), 724 - 30
Mouse palmitoyl protein thioesterase: gene structure and expression of cDNA; Salonen T et al.; Palmitoyl protein thioesterase (PPT) is the defective enzyme in infantile neuronal ceroid lipofuscinosis (INCL), which is a recessively inherited, progressive neurodegenerative disorder . We present here the cloning, chromosomal mapping, genomic structure, and the expression of the cDNA of mouse PPT . The mouse PPT gene spans >21 kb of genomic DNA and contains nine exons with a coding sequence of 918 bp . Fluorescence in situ hybridization to metaphase chromosomes localized the mouse PPT gene to the chromosome 4 conserved syntenic region with human chromosome 1p32 where the human PPT is located . PPT is expressed widely in a variety of mouse tissues . The mouse PPT cDNA is conserved highly with the human and rat PPT both at the nucleotide and amino acid sequence level . Transient expression of mouse PPT in COS-1 cells yielded a 38/36-kD differentially glycosylated polypeptide that was also secreted into culture media . Immunofluorescence analysis of transiently transfected HeLa cells indicated lysosomal localization of mouse PPT . Based on the high conservation of the gene and polypeptide structure as well as similar processing and intracellular localization, the function of PPT in mouse and human are likely to be very similar.

Mol Immunol, 1997 Dec, 34(18), 1273 - 80
Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component C1s; Tsai SW et al.; A 159 residue, N-terminal fragment of the human C1s complement component, C1s alpha(159), was expressed in the baculovirus, insect cell system . The protein was abundantly produced 3 days after infection, reaching levels as high as 40 microg/ml in cell culture media . It had a molecular weight of 18,100 (+/-4.9) Da by laser desorption mass spectrometry, close to the theoretical value of 18,111 Da, confirmed by sequencing . Sedimentation equilibrium and gel filtration column chromatography showed that C1s alpha(159) was a monomer in the presence of EDTA, and a dimer in the presence of Ca2+ . The C1s alpha(159)2 dimer had a sedimentation coefficient of 3.1 S . When the C1s alpha(159)2 was mixed with Clq, there was little or no interaction . Likewise, unactivated C1r2 dimer had a sedimentation coefficient of 6.8 S, and when mixed with C1q little or no interaction was observed . When C1s alpha(159)2 was mixed with the 6.8 S C1r2 in Ca2+, a 7.5 S complex was formed, presumably the C1s alpha(159) x C1r x C1r x C1s alpha(159) tetramer . When C1q, which migrated at 10.1 S was mixed with C1s alpha(159)2 and C1r2 in the presence of Ca2+, a C1-like complex, but containing C1s alpha(159) instead of C1s, was formed which migrated at 14.0 S . This C1-like molecule remained unactivated unless challenged with an ovalbumin-antiovalbumin immune complex . In the presence of immune complex, the C1r became activated . This suggested that the presence of the 159 amino acid C1s alpha domain, which held the C1r to the C1q, was sufficient to permit activation by an immune complex, even though the catalytic domains of C1s were not present.

Osteoarthritis Cartilage, 1998 May, 6(3), 196 - 204
Effects of chondroitin sulfate and interleukin-1 beta on human articular chondrocytes cultivated in clusters; Bassleer CT et al.; OBJECTIVE: To test the effects of chondroitin sulfate (ACS, a glycosaminoglycan of cartilage) with and without interleukin-1 beta (IL-1 beta) on human articular chondrocytes cultivated in clusters and in long-term (0-16 days or 16-32 days) . DESIGN: Chondrocyte productions of proteoglycans (PGs), type II collagen (coll-II) and prostaglandin E2 (PGE2) were assayed by specific radioimmunoassays applied to conditioned culture media and to clusters . RESULTS: During the two culture periods (0-16 days or 16-32 days), ACS (100-1000 micrograms/ml) increased total PG production and had no effect on the production of coll-II by chondrocytes . During the first 16 days, ACS (500-1000 micrograms/ml) decreased total PGE2 synthesis . IL-1 beta decreased PG and coll-II productions and increased PGE2 synthesis . During the first period (0-16 days), while the cluster is forming, ACS counteracted the IL-1 beta-induced effects on PG (500-1000 micrograms ACS/ml), coll-II (100-1000 micrograms ACS/ml) and PGE2 (500-1000 micrograms ACS/ml) productions . During the second period (16-32 days), when the cluster is already formed, ACS counteracted the IL-1 beta-induced effects on total PG (100-1000 micrograms ACS/ml), coll-II (1000 micrograms ACS/ml) and PGE2 (1000 micrograms ACS/ml) productions . CONCLUSION: These in vitro studies suggest that ACS is able to increase matrix component production by human chondrocytes and to inhibit the negative effects of IL-1 beta.

Exp Neurol, 1998 Jul, 152(1), 116 - 22
Role of peroxynitrite in the vasoactive and cytotoxic effects of Alzheimer's beta-amyloid1-40 peptide; Paris D et al.; Increasing evidence implicates oxidative stress as partially responsible for the neurodegenerative process of Alzheimer's disease (AD) . Recent reports show an increased production of nitrotyrosine in AD brains, suggesting that peroxynitrite is produced in excess in this disease . Furthermore, incidence of cerebral amyloid angiopathy in AD cases is very frequent (83%), strongly suggesting a vascular component of AD pathogenesis . We have evaluated the hypothesis that peroxynitrite could be responsible for mediating the cytotoxicity and vasoactivity induced by the amyloid-beta1-40 (Abeta) peptide . Rat brain endothelial cells (RBE-4) appear to be sensitive to Abeta-induced toxicity but not to the cytotoxicity induced by peroxynitrite . Addition of Cu/Zn superoxide dismutase to cell culture media, which is only able to clear extracellular superoxide, was not effective in blocking Abeta-induced toxicity . However, we were able to partially block Abeta-induced cytotoxicity by using Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) which dismutes superoxide intracellularily . Yet, MnTBAP was not able to prevent the vasoactivity triggered by Abeta . Moreover, addition of peroxynitrite to rat aortae did not modulate the vasotension induced by Abeta . We conclude that intracellular superoxide radicals may contribute to Abeta-induced cytotoxicity . Our results also indicate that peroxynitrite does not significantly contribute to Abeta-induced cytotoxicity in rat brain endothelial cells (RBE-4) or vasoactivity in rat aortae . These results suggest that therapeutic efforts aimed at removal of reactive oxygen species with SOD is unlikely to be beneficial for treatment of Abeta-induced endothelial dysfunction . However, compounds that clear free radicals intracellularly may well be beneficial .

Endocrinology, 1998 Aug, 139(8), 3597 - 605
The expression of interleukin-6 in the pregnant rat corpus luteum and its regulation by progesterone and glucocorticoid; Telleria CM et al.; Interleukin (IL)-6, a multifunctional cytokine originally described as a T cell-derived factor, is also produced by different cell types, and it influences a wide variety of physiological and pathophysiological processes . Recent studies further suggest that IL-6 has a role in down-regulating hormone production by endocrine organs and can negatively affect the steroidogenic capacity of both ovaries and testes . Thus, the aims of this investigation were to examine whether IL-6 plays a role in luteolysis and, more specifically, to determine whether luteal cells express the IL-6 gene, whether this expression is developmentally and hormonally regulated in pregnancy, and whether the corpus luteum could be a target for IL-6 action . Using semiquantitative RT-PCR, messenger RNA (mRNA) encoding both components of the IL-6 receptor {the ligand-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130)} were found to be highly expressed in corpora lutea throughout pregnancy . In contrast, IL-6 mRNA expression was barely detectable from day 4 through the end of pregnancy, whereas a sharp and abrupt expression of IL-6 mRNA occurred immediately after parturition . Although the corpus luteum does not express IL-6 mRNA during most of pregnancy, it could be induced to express this gene with an in vivo injection of the bacterial endotoxin, lipopolysaccharide . In addition, when corpora lutea from day-15 pregnant rats were isolated and maintained in culture, IL-6 mRNA that was undetectable at 0 h increased in a time-related manner and reached significant levels after 4 h of incubation, followed by a similar increase in IL-6 protein secreted in the culture media . Isolation of the small and large luteal cells by elutriation indicated that both cell populations can secrete IL-6 in culture . The apparent ability of luteal cells to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression during most of pregnancy, led us to examine whether the IL-6 gene is silenced throughout pregnancy by luteotropic hormones . Corpora lutea from day-15 pregnant rats were cultured in the presence of different doses of progesterone; the synthetic glucocorticoid, dexamethasone; 17beta-estradiol; and PRL . Progesterone and dexamethasone markedly inhibited IL-6 mRNA expression, whereas 17beta-estradiol had a minimal inhibitory effect, and PRL did not affect IL-6 mRNA expression . In summary, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and that luteal cells are able to secrete IL-6 . However, IL-6 gene expression is silenced during most of pregnancy, probably by the high levels of progesterone locally produced in the corpus luteum . The salient finding that progesterone and glucocorticoid strongly inhibit the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might have a deleterious effect on luteal function.

Curr Eye Res, 1998 Jul, 17(7), 694 - 9
Transferrin production by the ciliary body of rabbits: a biochemical and immunocytochemical study; Rodrigues ML et al.; PURPOSE: We have previously reported that transferrin is one of several glycoproteins synthesized within the eye and secreted into the vitreous . The present investigation was designed to determine the role of the ciliary body in the production of this vitreous transferrin . METHODS: Isolated ciliary body-iris were incubated with 3H-fucose, 3H-tyrosine or 35S-methionine and afterwards the culture media were processed for affinity chromatography using columns of Sepharose conjugated with antibody to rabbit plasma transferrin . Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from fresh ciliary body-iris and primers constructed on the basis of the known sequence of transferrin mRNA from rabbit liver . The fragment obtained was employed as a probe in northern-blots of total RNA of ciliary body-iris . Furthermore, paraffin sections of eyes were treated for immunocytochemical visualization of transferrin . RESULTS: A labeled polypeptide, specifically eluted from the antitransferrin columns, was detected in the incubation medium, transferrin mRNA was found in extracts of whole ciliary body-iris, and transferrin antigenicity was identified in the ciliary and iridial epithelial cells by immunocytochemistry . CONCLUSIONS: These results demonstrate the ciliary epithelium as one of the sources of the vitreous transferrin.

Skeletal Radiol, 1998 Jun, 27(6), 334 - 6
Considering Mycobacterium haemophilum in the differential diagnosis for lytic bone lesions in AIDS patients who present with ulcerating skin lesions; Lefkowitz RA et al.; Mycobacterium haemophilum has recently been recognized as a newly emerging cause of osteomyelitis in immunocompromised patients . While still uncommon, its incidence has increased significantly with the growing AIDS epidemic . Like its relative M . tuberculosis and M . intracellulare, this organism is acid-fast positive; yet unlike its more well-known counterparts, M . haemophilum requires iron-supplemented culture media and low incubation temperatures (30-32 degrees C) for growth . We describe a case of M . haemophilum osteomyelities in the distal femur of a 36-year-old HIV-positive male, who also presented with multiple skin ulcerations . In an AIDS patient with a lytic bone lesion and concomitant skin eruptions, the diagnosis of M . haemophilum should be entertained so that special culture media can be used and appropriate treatment administered.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 47 - 53
Multiplicity and expression of xylanases in the rumen fungus Neocallimastix frontalis; Gomez de Segura B et al.; The time course production of xylanolytic enzymes by the rumen anaerobic fungus Neocallimastix frontalis was studied during growth on different carbon sources and revealed using isoelectric focusing and immunoblotting . A constant low level of endoxylanase expression was observed in glucose medium . A high level of xylanase activity was detected in methyl glucoside medium corresponding to the induction of new isoforms which were repressed by the presence of glucose . beta-Xylosidases were constitutively produced at a high level and remained mainly associated to the fungal cells . Polyclonal antibodies raised against the endoxylanases XYLI and XYLII revealed that XYLI was secreted to the different culture media showing a characteristic pattern of constitutive expression, while anti-XYLII recognized several polypeptides larger than XYLII indicating the production of multiple antigenically related enzymes during growth on the inducing substrate.

Drugs Exp Clin Res, 1998, 24(2), 67 - 71
Suppressive effects of the new antirheumatic drug KE-298 on TNF alpha-induced production of matrix metalloproteinases but not of tissue inhibitor-1 of metalloproteinases in human rheumatoid synoviocytes; Takahashi S et al.; KE-298 is a novel antirheumatic drug which suppresses various animal models of arthritis by inhibiting the production of inflammatory cytokines . In a phase II study of rheumatoid arthritis patients, ingestion of KE-298 led to significant improvements in the Lansbury index . The objective of the present study was to clarify the effects of KE-298 against synovium functions, using rheumatoid arthritis synoviocytes . We investigated the effects of KE-298 on the production of matrix metalloproteinases and tissue inhibitor-1 of metalloproteinases and bone absorptive mediators including interleukin (IL)-6 and prostaglandin (PG) E2 in tumor necrosis factor (TNF)-alpha-stimulated rheumatoid arthritis synoviocytes . Rheumatoid arthritis synoviocytes were obtained from knee joints of rheumatoid arthritis patients and type B fibroblast-like synoviocytes were stimulated with TNF-alpha, with or without KE-298 . The contents of metalloproteinases and tissue inhibitor-1 of metalloproteinases and IL-6 and PGE2 in culture media were measured by enzyme-linked immunosorbent assay . KE-298 significantly suppressed TNF-alpha-induced production of promatrix metalloproteinase-1 and IL-6, in a dose-dependent manner, but not that of tissue inhibitor-1 of metalloproteinases . The potential of KE-298 to suppress the production of matrix metalloproteinase-1 and IL-6 may explain its efficacy on rheumatoid arthritis.

J Virol Methods, 1998 May, 72(1), 109 - 15
In vivo assay for hepatitis C viral serine protease activity using a secreted protein; Cho YG et al.; Hepatitis C virus (HCV) is a major pathogen of community-acquired and post-transfusional non-A, non-B hepatitis . Since an in vitro replication system is not available, it is crucial to develop an efficient and sensitive assay system for screening inhibitors of HCV . The fact that the activity of HCV NS3 protease is responsible for the maturation of the nonstructural proteins and viral replication, suggests that NS3 protease is a suitable target for anti-HCV drug development . To devise an assay system in cell culture, we constructed NS3/4A-SEAP (secreted alkaline phosphatase) chimeric gene, in which the SEAP gene was fused in-frame to downstream of NS4A/4B cleavage site . In this system, the SEAP would be secreted into the extracellular media depending on the cleavage activity of the NS3 protease . Our results demonstrate that the NS3/4A-SEAP expression vector encoding wild type NS3 protease, but not mutant NS3 protease, could produce high SEAP activity in the media of both transfected cells and stable expression cell lines . Since the activity of SEAP in the culture media can be monitored quantitatively and continuously by the chemiluminescent method, this assay system will be useful for screening potential inhibitors of HCV protease.

Mycoses, 1998 Mar-Apr, 41(3-4), 145 - 51
Demonstration of dermatophyte dissemination from the infected soles using the foot-press method; Maruyama R et al.; We have designed a new direct isolation method, the foot-press method, to survey dissemination of dermatophytes from the infected soles . A total of 56 untreated patients with tinea pedis were examined . The infected soles of 42 patients were pressed onto actidione-chloramphenicol-5-fluorocytosine (5FC)-gentamicin sulphate Sabouraud glucose medium prepared in a large culture dish; the culture media were then incubated at 25 degrees C . Dermatophytes were isolated in 30 out of the 42 patients, while no dermatophytes could be grown from 10 healthy controls . The number of isolated colonies from each patient ranged from 1 to 97 (mean +/- SD, 11 +/- 20) . The isolation frequencies were higher in the patients of hyperkeratotic type and in those with tinea unguium, while causative organisms and the extent of the lesions did not affect the results of the footpress method significantly . In order to reveal the morphology of disseminated dermatophytes, 1 x 1 cm pieces of culture media were cut out from culture dishes after pressing infected soles and were examined microscopically . Dermatophyte-like spores or hyphae, most of which were detached from cornified cells, could be seen in 10 out of 14 patients . Subsequently performed slide cultures isolated dermatophytes from approximately 70% of the pieces on which dermatophyte-like fungi were observed.

Anat Rec, 1998 Jul, 251(3), 303 - 15
Maternal antioxidant treatments prevent diabetes-induced alterations of mitochondrial morphology in rat embryos; Yang X et al.; Previous studies have suggested that production of reactive oxygen species by embryonic mitochondria may have a role in the induction of both high-amplitude mitochondrial swelling and embryonic dysmorphogenesis in diabetic pregnancy . The present study analyzed the relationships between a putative metabolite-induced production of free oxygen radicals, mitochondrial lipid peroxidation, and high-amplitude mitochondrial swelling in embryos during organogenesis . For studies in vitro, day 9 embryos of normal rats were cultured for 48 h with a high concentration of glucose in the absence or presence of alpha-cyano-4-hydroxycinnamic acid (CHC), a mitochondrial pyruvate transport inhibitor . The morphology of mitochondria in the neuroepithelium of the embryos was studied with the aid of transmission electron microscopy . For studies in vivo, normal and diabetic pregnant rats were fed a diet supplemented with the antioxidants alpha-tocopherol (vitamin E) or 2,6-di-tert-butyl-4-methylphenol (BHT), and the ultrastructure of mitochondria in the embryonic neuroepithelium and in the visceral yolk sac was investigated on gestational day 11 . Exposure to a high concentration of glucose in vitro or to maternal diabetes in vivo induced high-amplitude swelling of mitochondria in the neuroepithelium of the embryos . The swelling of mitochondria was prevented by addition of CHC to the culture media or by maternal ingestion of antioxidant-supplemented food . In diabetic pregnancy, embryonic mitochondria during organogenesis produce free oxygen radicals that cause mitochondrial lipid peroxidation and swelling and furthermore embryonic dysmorphogenesis . Dietary supplementation with antioxidants to the mother may prevent embryonic malformations in diabetic pregnancy by inhibition of mitochondrial dysfunction.

Mol Reprod Dev, 1998 Aug, 50(4), 434 - 42
Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids; Edwards LJ et al.; Although intracellular pH (pHi), is a regulator of numerous biological processes, it has received relatively little attention with regard to the physiology of the mammalian preimplantation embryo . Interestingly, there is some controversy as to whether the early embryo can recover from an acid load . The significance of this is that two constituents of mouse embryo culture media are pyruvate and lactate . These carboxylic acids are utilised by the early mouse embryo for energy production . However, as weak acids, pyruvate and lactate may induce perturbations in the pHi and thus alter the physiology of the embryo . The aims of this study were therefore to measure the pHi of the mouse preimplantation embryo and to determine the effect of lactate on pHi at different developmental stages . The pHi was measured using the ratio-metric fluorophore carboxy-seminaphthorhodafluor-1-acetoxymethylester (SNARF-1) in conjunction with confocal microscopy . The pHi increased significantly with development from the zygote to the morula stage . Furthermore, at concentrations greater than 5 mM, lactate caused the pHi of the zygote to become significantly more acidic . It was demonstrated that facilitative transport in association with a smaller passive component was responsible for the movement of lactate into the zygote . Metabolic studies revealed that, through their acidifying effect, weak acids caused a reduction in glycolytic activity in the early embryo . In contrast, the pHi of the compacted embryo remained unchanged by the presence of lactate in the external media . Furthermore, incubation with weak acids did not affect the rate of glycolysis in the morula . These data suggest that, by the generation of a transporting epithelium at compaction, the embryo develops the ability to regulate pHi against an acid load.

Ann N Y Acad Sci, 1998 Jun 29, 849, 307 - 12
Serum-free media for the in vitro cultivation of Cowdria ruminantium; Zweygarth E et al.; The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985; since then, most groups working with this culture system have used media which were supplemented with serum and, in addition, most of them contained tryptose phosphate broth . These undefined products vary from batch to batch and often fail to support the growth of C . ruminantium . We are therefore working towards the development of a completely chemically defined medium for Cowdria culture . We attempted the propagation of the Welgevonden stock of C . ruminantium in bovine endothelial cell cultures in a variety of serum-free culture media . Four synthetic media gave unsatisfactory results, these were: SFRE-199, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, and Leibovitz L-15 . These media were all supplemented with a proprietary solution A (components solution A of the HL-1 medium kit, containing transferrin, testosterone, sodium selenite, ethanolamine, saturated and unsaturated fatty acids, and stabilizing proteins) . Three other serum-free media did support the growth of C . ruminantium: a modified HL-1 medium, Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12), and RPMI 1640 . The chemical composition of DME/F-12 and RPMI 1640 are published, but not that of the HL-1 medium . Each of these media was supplemented with proprietary solution A . Various supplements were investigated as alternative to the incompletely specified solution A; bovine lipoproteins and bovine transferrin were identified as essential supplements which effectively replaced compound solution A . C . ruminantium was propagated in the three growth-supportive media for at least 10 passages.

Cytometry, 1998 Jul 1, 32(3), 223 - 32
In situ subcellular localization of epitope-tagged human and rabbit carboxylesterases; Potter PM et al.; Carboxylesterases are a ubiquitous class of enzymes thought to be involved in xenobiotic metabolism and detoxification . Primary amino acid sequence data suggest that these proteins localize to the endoplasmic reticulum . However, since this family of proteins is highly homologous, the generation of specific reagents to monitor expression and subcellular localization has been unsuccessful . To accomplish in situ detection of a human alveolar macrophage carboxylesterase and a rabbit liver carboxylesterase, we constructed plasmids that expressed recombinant proteins containing an 11 amino acid influenza hemagglutinin tag near the C-terminus . These proteins retained carboxylesterase activity as determined by the conversion of o-nitrophenol acetate to o-nitrophenol . Following transfection of plasmids encoding these proteins into mammalian cells, cells were analyzed by both fluorescence and electron microscopy . The tagged enzymes were localized to the endoplasmic reticulum of both Cos7 monkey kidney cells and Rh30 human rhabdomyosarcoma cells . No tagged protein was detectable in the culture media . Hence, epitope tagging allowed the analysis of expression and localization of specific carboxylesterases . The methods described in this paper are, therefore, applicable to any protein, including those that are highly homologous to other candidate molecules.

Br J Cancer, 1998 Jun, 77(11), 1744 - 51
Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes; Ortel B et al.; A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT) . Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes . Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA . These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells . The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media . Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged . From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific . Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation . Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation . Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.

Br J Urol, 1998 Jun, 81(6), 880 - 3
Temperature sensitivity of primary spermatocyte DNA synthesis in immature mice confirmed by bromodeoxyuridine labelling in vitro; Zhou B et al.; OBJECTIVE: To investigate the relationship between temperature and DNA synthesis of immature germ cells and to determine whether the early primary spermatocytes proliferate at a 'scrotal' temperature of 32 degrees C in vitro . MATERIALS AND METHODS: Day-7 mouse testes (n = 16) were cultured with 10% fetal calf serum (FCS) for 3 days at 32 degrees C or 37 degrees C and labelled by bromodeoxyuridine (BrDU) for a further hour . The BrDU-labelled cells were detected by immunohistochemical staining using a monoclonal anti-BrDU antibody . The numbers of primary spermatocytes with BrDU-labelling or unlabelled in each tubule were determined as an index of spermatocyte DNA synthesis . In addition, the cultured media at different temperatures were collected and the testosterone levels measured by radioimmunoassay . RESULTS: The numbers of primary spermatocytes (P < 0.01) and the BrDU-labelling index in primary spermatocytes per tubule at 32 degrees C in vitro were significantly higher (P < 0.001) than in the culture at 37 degrees C . The testosterone levels in the culture media at 32 degrees C were also markedly higher than in the culture at 37 degrees C (P < 0.01) . CONCLUSION: These observations indicate that DNA synthesis of early primary spermatocytes and testosterone production can be stimulated by lower testicular temperature, even in immature mouse testes that are naturally located in the intra-abdominal cavity.

Curr Microbiol, 1998 Aug, 37(2), 127 - 31
Metabolism of explosive compounds by sulfate-reducing bacteria; Boopathy R et al.; The metabolism of various explosive compounds-1,3,5-trinitrobenzene (TNB), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX)-by a sulfate-reducing bacterial consortium, Desulfovibrio spp., was studied . The results indicated that the Desulfovibrio spp . used all of the explosive compounds studied as their sole source of nitrogen for growth . The concentrations of TNB, RDX, and HMX in the culture media dropped to below the detection limit (<0.5 ppm) within 18 days of incubation . We also observed the production of ammonia from the nitro groups of the explosive compounds in the culture media . This ammonia served as a nitrogen source for the bacterial growth, and the concentration of ammonia later dropped to <0.5 mg/L . The sulfate-reducing bacteria may be useful in the anaerobic treatment of explosives-contaminated soil.

Bone, 1998 Jul, 23(1), 27 - 32
Mouse mammary carcinoma cell line (BALB/c-MC) stimulates osteoclast formation from mouse bone marrow cells through cell-to-cell contact; Ono K et al.; We recently reported that numerous osteoclasts (OC) were formed in cocultures of some mouse cancer cell lines and bone marrow cells . In this study, we examined mechanisms by which one of the cell lines, BALB/c-MC, induces OC . BALB/c-MC dose dependently stimulated OC formation in cocultures . In cocultures where direct cell-to-cell contact between BALB/c-MC and bone marrow cells was inhibited by membrane filters, OC formation was not stimulated . The stimulation of OC formation in the coculture was completely abolished by adding 10(-7)-10(-6) mol/L indomethacin . The concentration of prostaglandin E2 (PGE2) in the culture media of cocultures with cell-to-cell contact was higher than that of cocultures without cell-to-cell contact or marrow cultures alone, and it reached levels sufficient to induce OC (11.9 +/- 5.3 ng/mL {about 3.4 x 10(-8) mol/L}) . When BALB/c-MC or bone marrow cells were fixed with formalin and then cocultured with bone marrow cells or BALB/c-MC, respectively, the concentration of PGE2 in the culture media of cocultures of fixed BALB/c-MC and bone marrow cells increased, whereas that of cocultures of BALB/c-MC and fixed bone marrow cells did not increase . These results indicate that BALB/c-MC stimulate OC formation through direct cell-to-cell contact with bone marrow cells, and PGE2 released from bone marrow cells through direct cell-to-cell contact are involved in OC formation by the cell line.

Arch Insect Biochem Physiol, 1998, 38(3), 147 - 54
Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata; Komiya K et al.; The prothoracic glands (PGs) taken from the last instar of the common armyworm, Pseudaletia separata, were cultured in various media for the purpose of finding a suitable medium for relatively long-term culture of PGs . Among the tested culture media, MGM-450 medium without serum was the best to maintain PG cells viable for relatively long periods, and to continue to secrete ecdysteroids . Secretion of ecdysteroid by the PG in vitro became marked when the PG was taken from last instar larvae older than 2 days after the last molt . PGs cultured in any of the media secreted ecdysteroid only within the first 2 h after placing them in culture, however, in the MGM-450 medium, the PGs secreted ecdysteroid even after 5 days of culture.

Cardiovasc Res, 1998 Mar, 37(3), 820 - 5
Monocyte-vascular smooth muscle cell interaction enhances nitric oxide production; Ikeda U et al.; OBJECTIVE: The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis . We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression . METHODS: NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent . The expression of inducible NO synthase protein was assayed by Western blotting . RESULTS: Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner . The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner . Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation . Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes . Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha . The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody . CONCLUSIONS: The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.

J Dermatol Sci, 1998 Mar, 16(3), 200 - 7
Morphological, biochemical and molecular biological characteristics of a granulocyte colony-stimulating factor-producing human eccrine carcinoma cell line; Suga T et al.; We describe here a newly established cell line from an eccrine carcinoma which produced an abundant amount of granulocyte colony-stimulating factor (G-CSF) . An eccrine carcinoma of the scalp of a 69 year-old-Japanese female had metastasized to the pleura . Clinically, she had marked neutrophilia (up to 60,000/mm3), and a high level of G-CSF (38.7 x 10(3) pg/ml) was detected in the pleural effusion, as determined by enzyme-linked immunosorbent assay (ELISA) . We established a cell line in vitro and maintained the cells in culture for 30 months in 90 subcultures . We investigated whether these tumor cells were able to produce G-CSF in culture and found that they were . We also found that the amount of G-CSF produced paralleled the rise in cell number (26.5 x 10(3) pg/ml at confluency) . When culture media were administered to rabbits (25 ml/rabbit), the amount of circulating neutrophils increased until the number was equal to or greater than that resulting from injection of recombinant human G-CSF (rhG-CSF)(75 micrograms) . This effect persisted for 7 days . When tumors were induced in SCID and nude mice by injecting cultured cells (1 x 10(7) cells/mouse), the number of circulating neutrophils also correlated well with tumor size in these mice (200,000/mm3, 3 cm tumor) . After tumor removal, the neutrophil number returned to normal within 30 days . G-CSFmRNA in cultured, cells was detected by RT-PCR . Based on these results, it was confirmed that the marked neutrophilia observed in the patient was caused by the tumor-generated G-CSF . This is the first G-CSF-producing cell line developed from a cancer of the skin.

Eur J Clin Invest, 1998 May, 28(5), 424 - 9
Pneumocystis carinii induces interleukin 6 production by an alveolar epithelial cell line; Pottratz ST et al.; BACKGROUND: Pneumocystis carinii (PC) infection often results in severe pneumonia in immunocompromised patients . Attachment of PC organisms to alveolar epithelial cells is a hallmark of PC pneumonia; however, few studies have investigated the response of alveolar epithelial cells to PC infection . METHODS: Interleukin 6 (IL-6) is a multifunctional cytokine that is produced by alveolar epithelial cells in response to a variety of stimuli . Our investigation was undertaken to determine whether PC organisms induce production of IL-6 by alveolar epithelial cells and to determine the effect of IL-6 on PC attachment . RESULTS: Incubation of the human alveolar epithelial cell line, A549, with PC organisms resulted in a significant increase in IL-6 secreted into the cell culture media . Time-course studies showed that IL-6 production was detected as soon as 2 h after addition of PC and continued up to 48 h of exposure . Further studies demonstrated that preincubation of A549 cells with IL-6 resulted in a concentration-dependent increase in both A549 cell production of fibronectin and PC attachment . CONCLUSION: Thus, PC attachment to an alveolar epithelial cell line results in epithelial cell production of IL-6, which can act to further increase PC attachment . This may provide an important mechanism whereby PC organisms directly affect the host response to PC infection.

Circ Res, 1998 Jun 29, 82(12), 1338 - 48
Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor; Murasawa S et al.; The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects . In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts . Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation . Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation . Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function . Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187 . Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R . Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells . Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478 . Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.

Int J Impot Res, 1998 Jun, 10(2), 77 - 81
Development of an organ culture experimental system to investigate collagen synthesis in corpora cavernosa; Newhall PM et al.; An organ culture model of rabbit corpus cavernosum has been developed to investigate fibrillar collagen synthesis in intact organ . Rabbit peni were removed en bloc, the corpora cavernosa were dissected, sliced into 5 mm pieces and incubated in culture media up to 24 h . Tissue viability and metabolic and functional integrity were assessed by (a) lactate dehydrogenase (LDH) release, (b) protein synthesis, and (c) ability to respond to exogenously added cytokines . Explants, maintained for 24 h in organ culture, exhibited minimal LDH release and maintained functional integrity determined from protein synthesis and ability to synthesize collagen in response to TGF-beta(1) . These data suggest that explants of rabbit corpus cavernosum in organ culture represent a viable model to investigate connective tissue protein biosynthesis in vitro.

Hum Reprod, 1998 May, 13(5), 1312 - 6
Endogenous folic acid is essential for normal development of preimplantation embryos; O'Neill C; Preimplantation mammalian embryos develop with a high degree of autonomy . To date, there have been no unequivocal demonstrations of a requirement for vitamins in preimplantation embryo development . Reduced folic acid acts as an important methyl donor in many reactions including the synthesis of thymidine . Thymidine does not accumulate in cells so it might be expected that significant amounts of reduced folate would be required to support the exponential increase in DNA synthesis that occurs during early embryo development . The reduction of folate is catalysed by dihydrofolate reductase (EC 1.5.1.3) which is selectively inhibited by the anti-cancer drug methotrexate . Methotrexate caused a dose-dependent inhibition of cell division in 1-cell, 2-cell and 8-cell mouse embryos with 50% inhibition of division occurring at concentrations of 1-10 microM . At a concentration of 0.1 microM only minimal inhibition of the initial cell division occurred, but continuous culture in this concentration of methotrexate completely inhibited further cell divisions . This suggests that most of the exogenous store of reduced folates was used in the first round of cell division . The effects of methotrexate were apparently primarily due to thymidine starvation, since a 10-fold excess of thymidine over methotrexate in culture media reversed the inhibition of development . Supplementing media with folic acid had no beneficial effect on the rate at which zygotes produced by in-vitro fertilization developed to the blastocyst stage . It is concluded that the development of the early embryo has an absolute requirement for reduced folate for thymidine synthesis which is met entirely by endogenous sources.

Am J Reprod Immunol, 1998 Jun, 39(6), 356 - 61
Supplementation effect of early pregnancy factor-positive serum into bovine in vitro fertilization culture medium; Ito K et al.; PROBLEM: Early pregnancy factor (EPF) has been detected in pregnant bovine serum, and its activity appeared from 24 to 48 hr after insemination . However, in bovine in vitro fertilization (IVF), an EPF-like substance(s) had been detected in the culture medium of fertilized ovum . Therefore, we think that EPF and EPF-like substance(s) are very important materials for the development of the embryo . In this study, we examined the development of the embryo when fertilized bovine ova were cultured with IVF culture medium supplemented with EPF-positive or -negative serum . METHOD OF STUDY: EPF activity of each serum (fetal calf serum {FCS}, calf serum {CS}, estrus bovine serum, and pregnant bovine serum) was assessed by the bovine-rosette inhibition test . The sera were supplemented in TCM-199 culture medium, and IVF bovine ova were cultured with the media for 6 or 7 days, respectively . The culture media of each group were evaluated for EPF activity by the bovine-rosette inhibition test 48 hr after IVF . The cleavage rate of each group was calculated at 48 hr, and 6 or 7 days after IVF . The culture medium of cumulus cells was also tested for EPF activity . RESULTS: Only the pregnant bovine sera were EPF positive . All the culture media 48 hr after IVF became EPF positive . Additionally, the culture medium of cumulus cells did not have EPF activity . There was no significant difference in the cleavage rate of the EPF-positive and -negative sera 48 hr after IVF . However, the cleavage rate of EPF-positive sera tended to be higher than the negative sera . In contrast, the blastocyst development rates of EPF-positive sera were significantly higher than those of the negative sera 6 to 7 days after IVF (P < 0.05) . CONCLUSIONS: The data suggest that an EPF-like substance(s) may be secreted from the in vitro fertilized bovine ovum but not from the cumulus cell, and that the EPF in the pregnant serum may accelerate the development of the bovine embryo in IVF.

Am J Reprod Immunol, 1998 Jun, 39(6), 351 - 5
Early recognition of pregnancy by the maternal immune system; Kelemen K et al.; PROBLEM: Immunologic recognition of pregnancy is important for normal gestation . Successful pregnancy is characterized by a Th2 dominance, whereas there is a Th1 dominance in failed pregnancies . We assume that a signal given by the fertilized ovum induces a Th2 shift, necessary for a normal outcome . In vitro fertilization provides a tool for testing this possibility . METHOD OF STUDY: Phytohemagglutinin-stimulated lymphocytes were incubated for 48 hr in the presence of culture media from in vitro fertilized eggs, as well as in follicular fluid (FF) and control supernatants . Total RNA was isolated from the lymphocytes by the guanidine-isothiocyanate method and interleukin (IL)-10 mRNA expression was detected by reverse transcription-polymerase chain reaction . RESULTS: Ten percent of the activated lymphocytes incubated with FF expressed IL-10 mRNA, whereas 88% of the lymphocytes activated with supernatants of sperm + oocytes gave a positive signal . Significantly (P < 0.05) fewer (50%) lymphocytes stimulated in the presence of control supernatants also expressed mRNA for IL-10 . In supernatants of activated lymphocytes incubated with the culture medium of spermia + oocytes, the concentration of IL-10 was significantly higher than in the lymphocytes incubated with FF . CONCLUSION: These data suggest that the presence of the fertilized ovum induces a Th2 shift, which enables pregnancy to proceed to term.

Connect Tissue Res, 1998, 37(1-2), 87 - 103
Heparin sensitive and resistant vascular smooth muscle cells: biology and role in restenosis; San Antonio JD et al.; Vascular smooth muscle cells (VSMC)s are characterized by their acute growth inhibition by heparin and heparan sulfates; however, recently the isolation of VSMCs which display greatly diminished sensitivity to the antiproliferative action of heparin have been reported . These heparin resistant (HR) VSMCs have been derived through multiple passage of normal rat VSMCs in culture media containing high heparin doses, by transformation of VSMCs with oncogene-containing vectors, or have been isolated from vascular tissues of spontaneously hypertensive rats, healthy humans, or humans with restenosis where their presence is not limited to sites of injury . Initial characterizations of HR VSMCs are reviewed, and here we propose a definition of HR VSMCs . To date the mechanisms underlying heparin insensitivity remain elusive . Further study of HR VSMCs may expand our understanding of cell growth regulation by heparin, establish whether HR VSMCs contribute to the reported failure of heparin to combat restenosis in humans, and identify cellular mechanisms driving certain vascular proliferative diseases.

J Biomed Mater Res, 1998 Jul, 41(1), 87 - 94
Si-Ca-P xerogels and bone morphogenetic protein act synergistically on rat stromal marrow cell differentiation in vitro; Santos EM et al.; This study describes a novel bioactive xerogel glass as a carrier for bone morphogenetic protein (BMP) and the value of this carrier in terms of stimulating osteogenic activity of rat stromal marrow cells in vitro . These cells were seeded onto the surface of xerogel glass disks with BMP either incorporated in the glass, adsorbed to the surface of the glass, or added to the culture media and then compared to cells on glass with no added BMP or to cells on tissue culture plastic (TCP) with and without BMP . Cells were cultured for 6 and 10 days and examined for total DNA, alkaline phosphatase activity, and osteocalcin and total protein production . Stromal cell differentiation, as measured by alkaline phosphatase activity and osteocalcin synthesis was most increased when the BMP was incorporated or adsorbed onto the xerogel glass . Cells on xerogel glass without BMP were more differentiated than cells grown on plastic with BMP, thereby demonstrating the additive effect of a bioactive substrate and BMP on osteoblastic cell differentiation . These data indicate that xerogel glass effects differentiation of cells with osteogenic potential and that it can serve as a delivery vehicle for BMP.

J Atheroscler Thromb, 1997, 4(2), 79 - 84
Effects of copper-zinc type superoxide dismutase on the proliferation and migration of cultured vascular smooth muscle cells induced by oxidized low density lipoprotein; Oinuma T et al.; We have investigated the effects of copper-zinc type superoxide dismutase (Cu, Zn-SOD) on the function of oxidized low density lipoprotein, utilizing cultured smooth muscle cells (SMC), obtained from rabbit aorta . We added native LDL (nLDL), minimally oxidized LDL (MmLDL) and copper ion-induced oxidized LDL (OxLDL) to the culture media . No remarkable change was found out by adding nLDL . The numbers of SMC, including migrated SMC, were increased by the addition of MmLDL . Cu, Zn-SOD significantly inhibited the reactions induced by MmLDL . The SMC numbers were markedly decreased by OxLDL addition without recovery by adding Cu, Zn-SOD . Thus, MmLDL significantly promoted the SMC proliferation and migration . OxLDL revealed strong cytotoxicity against SMC . Cu, Zn-SOD inhibited both the migration and the proliferation of SMC induced by MmLDL, and did not alter the effect of OxLDL . In conclusion, Cu, Zn-SOD inhibited some functions of MmLDL, and may play an important role in protecting against the atherosclerotic processes evoked by MmLDL.

Vet J, 1998 May, 155(3), 231 - 7
Isolation and identification of canine matrix metalloproteinase-2 (MMP-2); Coughlan AR et al.; A canine gelatinase, with an apparent molecular mass of 62 kDa in non-reducing zymography, is produced by fibroblasts, chondrocytes and a myelomonocytic cell line . The enzyme has similar characteristics to human matrix metalloproteinase (MMP) 2 and cross-reacts in Western blotting analysis with a sheep polyclonal antiserum raised against human MMP-2 . The 62 kDa canine protein was purified from cell culture media, and the N-terminal amino acid sequence determined following blotting on to a polyvinylidene difluoride (PVDF) membrane . The sequence was 87% identical to that published for human MMP-2 . We therefore consider this enzyme to be canine MMP-2.

J Clin Invest, 1998 Jun 15, 101(12), 2821 - 30
Characterization of a CD38-like 78-kilodalton soluble protein released from B cell lines derived from patients with X-linked agammaglobulinemia; Mallone R et al.; Studies on murine B lymphocytes showed that Bruton's tyrosine kinase mediates signal transduction induced via CD38, a nonlineage-restricted 45-kD ectoenzyme . This signaling is defective in B cells from X-linked immunodeficient mice affected with the analogue of human X-linked agammaglobulinemia (XLA) . We performed a structural and functional analysis of CD38 in XLA and other immunodeficiencies, using EBV-immortalized B cells derived from such patients . Membrane CD38 was not significantly different from controls in structure, epitope density, enzymatic activity, and internalization upon binding of agonistic mAbs . Meanwhile, an increased release of soluble CD38 from XLA cells was observed: immunoprecipitation from XLA culture media yielded a protein of approximately 78 kD (p78), reacting also in Western blot and displaying both enzymatic activities and a peptide map similar to membrane CD38 . Soluble forms and homotypic aggregations of CD38 were documented in different cell models and by crystallographic analysis of the Aplysia ADP-ribosyl cyclase, the ancestor of human CD38 . p78 might represent the product of an altered turn-over of membrane CD38, a starting point for studying its association with Bruton's tyrosine kinase and its role in XLA and other B cell immunodeficiencies.

Arterioscler Thromb Vasc Biol, 1998 Jun, 18(6), 984 - 90
Dominant negative effect of TGF-beta1 and TNF-alpha on basal and IL-6-induced lipoprotein(a) and apolipoprotein(a) mRNA expression in primary monkey hepatocyte cultures; Ramharack R et al.; Lipoprotein(a) {Lp(a)} consists of apolipoprotein(a) {apo(a)} disulfide linked to apolipoprotein B-100 of LDL . Elevated plasma Lp(a) is an independent risk factor for a variety of vascular diseases . Lp(a) has been reported to be an acute-phase reactant, suggesting that cytokines may regulate its levels . To determine whether Lp(a) expression was subject to modulation by cytokines, primary monkey hepatocytes that endogenously express Lp(a) were used . Hepatocytes were treated with interleukin (IL)-6, the major mediator of the acute-phase response, and several other cytokines . IL-6 treatment (0.3 to 10 ng/mL) resulted in a marked, dose-dependent, 2- to 4-fold enhancement of Lp(a) accumulation in the hepatocyte culture media that was highly correlated with changes in apo(a) mRNA levels (r>0.9) . Several other cytokines, such as IL-2, IL-8, and hepatocyte growth factor, had no significant effect on Lp(a) levels; however, transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha) were very active in inhibiting Lp(a) accumulation in the culture media, with IC50s of approximately 0.3 and 1 ng/mL, respectively . Both TGF-beta1 and TNF-alpha also decreased the apo(a) transcript . Mixing experiments, in which hepatocytes were treated with 10 ng/mL of IL-6 and 0.3 to 10 ng/mL of TGF-beta1 or TNF-alpha, demonstrated that the IL-6-mediated induction of Lp(a) and apo(a) mRNA was ablated with very low levels of either inhibitory cytokine, suggesting a dominant negative effect of TGF-beta1 and TNF-alpha . These results show that Lp(a) and apo(a) mRNA expression in primary monkey hepatocytes is subject to both positive (IL-6) and negative (TGF-beta1 and TNF-alpha) regulation by physiological levels of cytokines . Thus, in vivo Lp(a) levels may be dependent on the balance between stimulatory and inhibitory cytokines.

J Neurosci Res, 1998 Jun 1, 52(5), 521 - 9
Potassium current in Drosophila neurons is increased by either dunce mutation or cyclic AMP; Alshuaib WB et al.; In the Drosophila mutant dunce, short-term memory is deficient and intracellular cyclic adenosine monophosphate (cAMP) concentration is elevated . We examined the effect of increased cAMP concentration on the potassium current . The conventional whole-cell technique was applied to cultured "giant" Drosophila neurons derived from cell-division arrested embryonic neuroblasts . Potassium membrane currents were measured from: 1) control wild-type neurons, 2) wild-type neurons with dibutyryl cAMP and theophylline in the culture media for 2 days (db-cAMP-treated), and 3) dunce neurons . Delayed-rectifier potassium current was greater in both dunce neurons and db-cAMP-treated wild-type neurons than in control wild-type neurons . This result indicates that the neuronal potassium current is increased by the long-term increase of cAMP . Conceivably, altered neuronal excitability in the dunce mutant could disrupt the processing of neural signals necessary for learning and memory.

Exp Eye Res, 1998 May, 66(5), 513 - 9
Mucin-like glycoprotein secretion is mediated by cyclic-AMP and protein kinase C signal transduction pathways in rat corneal epithelium; Nakamura M et al.; Ocular surface mucin is secreted from both goblet cells in the conjunctival epithelium and corneal epithelial cells . To clarify its mechanism of secretion in corneal epithelial cells, a rat cornea organ culture system was used to evaluate the second messenger roles of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and protein kinase C (PKC) in modulating mucin-like glycoprotein secretion . Rat cornea sections (3 mm diameter) were cultured in TC-199 medium, and radiolabeled with sodium sulfate for 18 hr . After washing, the corneas were treated with various second messenger modulating agents for 30 min . The culture media were reacted with Dolichos biflorus (DBA)-lectin, and mucin-like glycoprotein was isolated . Then the radioactivity of DBA-binding mucin-like glycoprotein was isolated . Then the radioactivity of DBA-binding mucin-like glycoprotein was measured . There was a time-dependent increase in mucin-like glycoprotein was measured . There was a time-dependent increase in mucin-like glycoprotein secretion, whereas after corneal epithelial debridement the secretion was markedly inhibited by 81% . Mucin-like glycoprotein secretion was stimulated in a dose-dependent manner following elevation of cAMP levels by exposure to either forskolin, dibutyryl cAMP or 3-isobutyl-1-methylxanthine . Concomitant exposure to the cAMP dependent protein kinase inhibitor, KT5720 completely inhibited their stimulatory effects . Neither exposure to dibutyryl cGMP nor nitroprusside affected mucin-like glycoprotein secretion . Stimulation by PKC, phorbol 12, 13-dibutyrate (PDBu) also increased mucin-like glycoprotein secretion in a dose-dependent fashion . The PKC inhibitor, calphostin C completely inhibited the stimulation by PDBu of mucine-like glycoprotein secretion . These results demonstrate that corneal epithelial cells secrete mucin-like glycoprotein, which is mediated by cAMP and PKC signal transduction pathways.

Neurosci Lett, 1998 Apr 24, 246(2), 120 - 2
In vitro changes in capacitative Ca2+ entry in neuroblastoma X glioma NG108-15 cells; Ichikawa J et al.; Changes in capacitative Ca2+ entry were studied in neuroblastoma x glioma NG108-15 cells with fura-2 fluorescence measurements in the following three culture conditions . The application of thapsigargin (250 nM) with a Ca2+-free solution depleted intracellular Ca2+ stores and the capacitative Ca2+ entry was induced by the addition of extracellular Ca2+ in the cells cultured in the medium for proliferation . The capacitative Ca2+ entry decreased in the cells cultured in the medium for neuronal differentiation . When these cells resumed proliferation after changing the culture media to the initial medium for proliferation, the capacitative Ca2+ entry increased again and exceeded the level in the initial proliferation state . These results suggested that the capacitative Ca2+ entry occurred more intensely at the proliferation state than at the neuronally differentiated state.

Arthritis Rheum, 1998 Jun, 41(6), 997 - 1006
Regulation of cartilage oligomeric matrix protein synthesis in human synovial cells and articular chondrocytes; Recklies AD et al.; OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a component of the extracellular matrix of articular cartilage . Its increased presence in synovial fluid and serum has been associated with accelerated joint damage in patients with rheumatoid arthritis (RA) and osteoarthritis . To fully understand the reasons for fluctuations of COMP levels, we studied the biosynthesis of this molecule in cells derived from joint tissues . METHODS: Synovial cells were derived from synovial tissues of patients with RA, and human articular chondrocytes were prepared from normal articular cartilage . Analysis by Northern blotting was used to evaluate steady-state levels of COMP messenger RNA (mRNA), while secretion of the protein into culture media was analyzed by Western blotting . Expression of COMP in synovial tissues was studied by reverse transcriptase-polymerase chain reaction analysis and by in situ hybridization . RESULTS: COMP was synthesized and secreted by synovial cells as well as by articular chondrocytes in culture . The basal rate of synthesis was very low; however, COMP biosynthesis in both cell populations was induced very strongly by transforming growth factor beta1 (TGFbeta1) . Interleukin-1beta counteracted COMP induction by TGF-beta1 . COMP was not detected in culture media of skin or fetal lung fibroblasts, either in the absence or the presence of TGFbeta1 . COMP mRNA was also present in fresh synovial tissue specimens obtained from patients with RA . CONCLUSION: COMP is synthesized and secreted not only by articular chondrocytes, but also by synovial fibroblasts . The demonstration of COMP expression in surgical specimens of synovial tissues suggests that the inflamed synovium may provide an additional source for the elevated levels of COMP observed in arthritis . Thus, increased COMP levels in body fluids may be indicative of active synovitis as well as of accelerated joint erosion.

Brain Res, 1998 Apr 20, 790(1-2), 151 - 9
Neurally mediated hyperactive voiding in spontaneously hypertensive rats; Spitsbergen JM et al.; The development of hypertension in spontaneously hypertensive rats (SHR) and hyperactive voiding in rats with urethral obstruction are characterized by abnormal smooth muscle growth, increased tissue levels of nerve growth factor (NGF) and altered patterns of innervation . The present study was undertaken to determine if bladder smooth muscle from SHRs contains and secretes elevated levels of NGF, and if so, whether the augmented NGF contributes to changes in bladder innervation and function without tissue hypertrophy . Voiding behavior was monitored using specially designed metabolic cages . NGF levels in tissue homogenates and conditioned cell culture media were measured by ELISA . NGF mRNA in cultured bladder smooth muscle cells (BSMCs) was quantified using reverse transcriptase PCR . Noradrenergic innervation was assessed by staining with glyoxylic acid and assaying norepinephrine (NE) content in bladders with high performance liquid chromatography . SHRs voided more frequently than WKY rats . NGF content was higher in bladders from adult SHRs when compared to Wistar-Kyoto normotensive rats (WKYs) . No significant difference in NGF mRNA content was observed between SHR and WKY BSMCs . However, SHR BSMCs secreted NGF at a higher rate and amount per unit mRNA than did WKY BSMCs . SHR bladders contained more NE and were more densely stained for catecholaminergic fibers than bladders from WKY rats . The results support the hypothesis that elevated NGF secretion by bladder smooth muscle is associated with hyperinnervation of bladder and hyperactive voiding in SHRs . Thus, the SHR strain may represent a genetic model to study changes in bladder function resulting from altered patterns of innervation .

Biochim Biophys Acta, 1998 Mar 5, 1406(2), 169 - 74
17beta-oestradiol enhances release of matrix metalloproteinase-2 from human vascular smooth muscle cells; Wingrove CS et al.; Vascular remodelling occurs during all stages of atherosclerotic progression . Anti-atherosclerotic drugs may function by restoring regulation of the processes involved in remodelling of the extracellular matrix . A key group of enzymes involved in these processes are the matrix metalloproteinases (MMPs) . Oestrogens have been demonstrated to possess anti-atherosclerotic properties at low concentrations while being associated with lesion formation at high concentrations . We examined the effect of 17beta-oestradiol on MMP-2 expression in human coronary artery (CAVSMC) and umbilical artery vascular smooth muscle cells (UAVSMC) . MMP-2 expression was measured by chemiluminescent immunoblotting and quantified by laser densitometry . pro-MMP-2 was secreted by VSMCs and increasing levels of 17beta-oestradiol, from physiological through supraphysiological, were associated with significant dose-dependent increases in MMP-2 levels in culture media . This effect was dependent on de novo protein synthesis and could be antagonised by the oestrogen receptor antagonist, tamoxifen, and the specific receptor antagonist ICI 182, 780 . 17beta-Oestradiol appears to be a specific stimulator of MMP-2 release from human vascular cells . The concentration dependence of this effect suggests a basis for the differential effects of low and high oestrogen levels on vascular integrity .

J Biol Chem, 1998 May 8, 273(19), 11806 - 14
Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type; Wei L et al.; A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I) . Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes . Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media . While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein . Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C . Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion . However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients . Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation.

Shock, 1998 Apr, 9(4), 266 - 73
8-ISO-PGF2alpha production by alveolar macrophages exposed to hyperoxia; Vacchiano CA et al.; Oxygen exposure for a sufficient duration at high partial pressure results in pulmonary edema in humans and animals . Although the specific mediators of oxygen toxicity are unknown, evidence suggests that oxygen-based radicals such as superoxide anion (O2.) are increased in the lungs in the presence of hyperoxia and contribute to this injury . A series of isomeric prostanoid compounds, the isoprostanes, are formed by the free radical-initiated lipid peroxidation of arachidonic acid (AA) . One of these isomers, 8-iso-PGF2alpha, is elevated in the bronchial alveolar lavage fluid of rats exposed to 90% oxygen for 48 h and is associated with a significant increase in protein accumulation in the pulmonary extravascular space . Alveolar macrophages (AMs) are capable of producing large quantities of (O2.), suggesting a role in pulmonary oxygen toxicity . We hypothesized that isolated rat AMs exposed to hyperoxia generate increased amount of 8-iso-PGF2alpha . AMs were exposed to air or 90% oxygen for 6, 12, 24, 48, 72, 96, and 120 h in the absence and presence of AA and/or calcium ionophore (A23187) and 8-iso-PGF2alpha was measured in the culture media . Exposure of primary cultures of AMs to 90% oxygen resulted in a significant increase in 8-iso-PGF2alpha in the media (25 +/- 2 pg/mL) compared with air-exposed controls (14 +/- 1 pg/mL) . The addition of 10 microM AA and 2 microM A23187 to the culture media resulted in a marked increase in 8-iso-PGF2alpha production by AMs exposed to air and 90% oxygen . However, treatment of AMs with the combination of AA and A23187, followed by exposure to 90% oxygen for 72 h, resulted in a 27-fold increase in 8-iso-PGF2alpha compared with media alone and 90% oxygen . AMs metabolized free and phospholipid-bound AA to 8-iso-PGF2alpha, an activity enhanced in the 90% oxygen environment . Finally, acetylsalicylic acid, a cyclooxygenase inhibitor and free radical scavenger, reduced but did not abolish production of 8-iso-PGF2alpha . This study provides evidence that AMs produce a free radical-mediated isomeric prostaglandin compound that may be involved in pulmonary oxygen toxicity.

Biochim Biophys Acta, 1998 May 27, 1403(1), 37 - 46
Local control of alpha1-proteinase inhibitor levels: regulation of alpha1-proteinase inhibitor in the human cornea by growth factors and cytokines; Boskovic G et al.; Alpha 1-proteinase inhibitor is a major serine proteinase inhibitor in the human cornea involved in the protection of the avascular corneal tissue against proteolytic damage . This inhibitor is upregulated systemically during infection, inflammation and injury . Cytokines that mediate the acute phase response such as IL-1beta and IL-2 increased alpha1-proteinase inhibitor present in corneal organ culture media . This released inhibitor represented mainly newly synthesized protein . However, IL-6, a general inducer of the acute phase response that upregulates alpha1-proteinase inhibitor in all other tissues and cells tested, failed to alter corneal alpha1-proteinase inhibitor levels over the tested period of 24 h . In addition to IL-1beta and IL-2, alpha1-proteinase inhibitor levels in the corneal organ culture medium increased following the addition of FGF-2 and IGF-I . The effect of the above growth factors and cytokines was relatively fast with maximal induction observed within the first 5 h . Among the tested growth factors and cytokines, IL-1beta was the most potent and increased total corneal alpha1-proteinase inhibitor levels approximately 2.4-fold in the cornea organ culture medium . Newly, synthesized alpha1-proteinase secreted into the medium increased 3.9-fold . In addition to the effect on corneal alpha1-proteinase inhibitor, IL-1beta also increased the amount of alpha1-proteinase inhibitor released by monocytes and macrophages but not by HepG2, CaCo2, and MCF-7 cells within 24 h . These results suggest that the cornea can locally control levels of alpha1-proteinase inhibitor in response to an inflammatory insult .

J Cell Biochem, 1998 Jun 15, 69(4), 453 - 62
Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation; Nie D et al.; Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification . To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization . The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology . The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin . RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells . There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes . RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells . Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix . These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization . They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification.

J Cancer Res Clin Oncol, 1998, 124(3-4), 155 - 64
The in vitro influence of eight hormones and growth factors on the proliferation of eight sarcoma cell lines; Remmelink M et al.; Little is known about the regulation of sarcoma proliferation by hormones and/or growth factors . We therefore characterised the in vitro proliferative influence on eight sarcoma cell lines of the platelet-derived growth factor, the insulin-like growth factor 1, triiodothyronine, the epidermal growth factor, the luteinising-hormone-releasing hormone, progesterone, gastrin and 17 beta-oestradiol . The influence of the different factors on the proliferation of sarcoma cell lines was measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test . Two culture media were studied: (1) a nutritionally poor medium containing 2% of fetal calf serum and (2) a nutritionally rich one containing 5% or 10% FCS both with and without the addition of non-essential amino acids . The results were analysed either by conventional statistical analyses or by a classification method based on a decision-tree approach developed in Machine Learning . This latter method was also compared to other classifiers (such as logistic regression and k nearest neighbours) with respect to its accuracy of classification . Monovariate statistical analysis showed that each of the eight cell lines exhibited sensitivity to at least one factor, and each factor significantly modified the proliferation of five or six of the eight cell lines under study . Of these eight lines one of fibrosarcoma origin was the most "factor-sensitive" . Decision-tree-related data analysis enabled the specific pattern of factor sensitivity to be characterised for the three histological types of cell line under study . The effects of hormone and growth factors are significantly influenced by the type of culture medium used . The method used appeared to be an accurate classifier for the kind of data analysed . Sarcoma proliferation can be modulated, at least in vitro, by various hormones and growth factors, and the proliferation of each histopathological type exhibited a distinct sensitivity to different hormone and/or growth-factors.

Hum Reprod, 1998 Apr, 13(4), 998 - 1002
Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos; Yang HW et al.; In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media . ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells . This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2 . A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme . The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit . The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05) . Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos . Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres . We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.

Hum Reprod, 1998 Apr, 13(4), 927 - 35
Inhibin A and B secretion in mouse preantral follicle culture; Smitz J et al.; Conditioned media from single mouse ovarian follicles cultured from the early preantral stage up to complete maturity were analysed for different immunoreactive inhibin forms . The inhibin assays measured (i) alpha-specific inhibin, as represented by a mix of 32 and and 57 kDa inhibins, inhibin precursors, alpha-subunit and its precursors; (ii) dimeric inhibin A; and (iii) dimeric inhibin B . The validity of these assays for the measurement of mouse inhibin was established . All forms of inhibin were secreted in culture media from the preantral follicle stage onwards . Inhibin B was the most sensitive marker for proliferation of early stage follicles, while inhibin A secretion became predominant at later stages, when antral-like cavities were formed in granulosa cell masses . Supplementation of standard culture medium with recombinant follicle stimulating hormone appeared to be the predominant regulator of inhibin secretion; addition of recombinant luteinizing hormone throughout the culture period did not cause any major shifts in the expression of dimeric inhibin or alpha-specific inhibin forms . In the absence of theca cells during isolation and culture (as reflected by absence of oestrogen secretion), follicles grew at a reduced rate, and produced lower inhibin concentration in conditioned medium . These data suggest (i) that monitoring of dimeric inhibins can provide useful markers of the growth and differentiation of cultured follicles and (ii) that dimeric inhibins A and B are secreted at an earlier stage in vitro than in vivo.

FASEB J, 1998 Jun, 12(9), 747 - 52
High expression of leptin by human bone marrow adipocytes in primary culture; Laharrague P et al.; Adipocytes participate in the microenvironment of the bone marrow (BM), but their exact role remains to be determined . It has recently been shown that leptin, a hormone secreted from extramedullary adipocytes, could be involved in hematopoiesis . Therefore we have developed a primary culture system of human BM adipocytes to characterize their differentiation and determine whether leptin is also secreted from these adipocytes . BM cells were cultured with fetal calf and horse sera . In the presence of dexamethasone, cells with vesicles containing lipids appeared within 15 days . They expressed glycerol phosphate dehydrogenase activity and a lipolytic activity in response to isoproterenol, but expressed neither the adrenergic beta3 receptor nor the mitochondrial uncoupling protein UCP1 . The addition of insulin alone to the culture media did not promote adipocyte differentiation . Leptin was expressed and secreted at high levels during adipocyte differentiation . Acute exposure of differentiated adipocytes to insulin had little effect on leptin expression whereas forskolin strongly inhibited it . These results show that although human BM adipocytes differ from extramedullary adipose tissues in their sensitivity to different effectors, they are a secondary source of leptin production . They suggest that BM adipocytes could contribute to hematopoiesis via the secretion of leptin in the vicinity of hematopoietic stem cells.

Steroids, 1998 May-Jun, 63(5-6), 285 - 7
Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP); Ducray A et al.; The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP) . As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones . The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures . Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks . Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP . Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector . Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.

J Cell Physiol, 1998 Jul, 176(1), 166 - 78
Determinants of glutamine dependence and utilization by normal and tumor-derived breast cell lines; Collins CL et al.; A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth . GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media . However, the GLN requirements of tumor cell lines and the factors that determine a cell's need for GLN have not been comprehensively studied . Also, it remains unclear how various metabolic pathways contribute to GLN consumption . In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin . These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates . GLN uptake was facilitated by a single, common transporter functionally defined as System ASC . However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines . Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity . The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor-derived cells . The rates of CO2 production from GLN were similar for each cell lines . Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32-80% of media GLN converted to glutamate . Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system x(c)- . These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis.

Intern Med, 1998 Mar, 37(3), 253 - 8
Correlations between interleukin-8, and myeloperoxidase or luminol-dependent chemiluminescence in inflamed mucosa of ulcerative colitis; Anezaki K et al.; Interleukin-8 (IL-8) is a peptide which induces not only chemotaxis of neutrophils but also the release of reactive oxygen metabolites from the neutrophils . There are few reports which clarify the relationships between IL-8 and mucosal infiltration of neutrophils or reactive oxygen metabolites produced by neutrophils in the colonic mucosa of ulcerative colitis (UC) . Biopsy specimens of colonic mucosa obtained from 26 patients with active UC and 21 patients with inactive UC were studied in order to clarify the relationships among the inflammation factors in UC . Levels of IL-8 and myeloperoxidase in organ culture media of the biopsy specimens from active UC (measured by ELISA and EIA) were significantly higher than those from inactive UC and controls . Reactive oxygen metabolites of biopsy specimens in active UC (measured by luminol-dependent chemiluminescence) were also markedly increased compared to those in inactive UC and controls . The levels of IL-8 were closely correlated to luminol-dependent chemiluminescence or myeloperoxidase levels . However, the levels of IL-8 and myeloperoxidase did not correlate with the grades of activity on colonoendoscopic findings . These findings suggest that IL-8 may play a role in the pathophysiology of UC but it does not define the endoscopic activity grades of UC.

J Immunol Methods, 1998 Feb 1, 211(1-2), 9 - 20
Detection of thioredoxin in human serum and biological samples using a sensitive sandwich ELISA with digoxigenin-labeled antibody; Das KC et al.; Thioredoxin is a low molecular weight, redox active protein important in cellular proliferation, signal transduction and antioxidant function . Thioredoxin is secreted by normal as well as neoplastic cells and is potentially involved in paracrine cell communication as suggested by its co-cytokine activity . Thus, the thioredoxin level in biological fluids, cells and tissue homogenates could be an important indicator of physiological or pathophysiological conditions . Hence, an accurate and sensitive measurement is of paramount importance in studies involving thioredoxin . We present here an ultrasensitive enzyme linked immuno-absorbent assay (ELISA) for human thioredoxin using digoxigenin-labelled goat polyclonal anti-human thioredoxin . The assay could detect a minimum level of 15 pg/ml thioredoxin in human serum, cell culture media, and in cell and tissue samples . The assay was optimized for concentration of both antibodies, blocking agent, plates, incubation time and reaction volumes . Excellent linearity and reproducibility were obtained . The assay was applied to different baboon tissues and human serum samples . The intrassay coefficient of variation (CV) was between 6.0 to 14 and the interassay CV was from 1.6 to 11.1 . Excellent parallelism of standards with serum samples, tissue homogenates or cell lysates was obtained . More than 90% recovery of human thioredoxin was observed in 10% human serum . The assay is easy to use, rapid, reproducible, but above all it is a quantitative, specific and sensitive way to measure thioredoxin in a variety of biological specimens.

Brain Res, 1998 Mar 2, 785(2), 253 - 61
ETA and ETB receptor antagonists synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures; Hasselblatt M et al.; Astrocytes produce and bind endothelins (ETs), suggesting that these cells have ET autoregulatory and eliminatory functions . To further investigate these functions in primary rat astrocytes, ET-1 levels in the cell culture media (RIA/HPLC) and intracellular content of ET-1 mRNA (RT PCR) were measured under basal and stimulated (thrombin, 2.2 U/ml) conditions in the presence and absence of ETA and ETB selective antagonists (BQ123 or LU135252, and BQ788, respectively) . Neither basal nor stimulated ET-1 levels in astrocyte media were influenced by ETA or ETB antagonists alone, but were significantly increased by a combination of both . ir ET-3 levels were not affected by antagonist treatment . Exogenous ET-1, added to the cultures, was rapidly cleared from the supernatant; this clearance was markedly inhibited by a combination of BQ123 and BQ788 . ET-1 mRNA levels were not altered by any treatment . To conclude, in primary rat astrocyte cultures, extracellular ET-1 is cleared by binding to ET-receptors, apparently involving both, ETA and ETB sites . Thus, a blockade of the astrocytic ET eliminatory function as a consequence of the in vivo application of non-selective ET receptor antagonists may lead to increased extracellular ET levels in the brain .

Vet Immunol Immunopathol, 1998 Feb 16, 61(1), 1 - 16
Studies on canine bone marrow long-term culture: effect of stem cell factor; Neuner E et al.; Long-term culture of canine marrow cells allows in vitro studies of the hematopoietic system of the dog and characterization of early progenitor cells . Colonies of fresh marrow cells grew equally good in both agar or methylcellulose supplemented with fetal calf serum, while colonies of long-term cultures required agar-based medium containing human serum . Optimum colony growth was obtained when stem cell factor (SCF) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) were used as growth stimuli of colony forming units (CFU) . Similar results were achieved with several cell culture media . Addition of hydrocortisone to long-term cultures improved clonogenic growth of cultured cells . Addition of 2-mercaptoethanol had no effect . Strong differences were observed in long-term culture with different horse serum lots and the addition of fetal calf serum to long-term culture suppressed CFU growth of cultured cells . Recharging of cultures with fresh marrow cells on day 7 of culture improved CFU growth only in the following week but had little effect on the outcome . Adding SCF to long-term cultures led to differentiation of more primitive cells and destruction of the stromal layer . Investigation of purified and cultured cell populations was possible when preestablished long-term cultures as stromal layers were used . Loss of long-term culture-initiating ability could be demonstrated in this system with lineage negative marrow cells expanded ex vivo with SCF and GM-CSF.

Eur J Gynaecol Oncol, 1998, 19(2), 113 - 9
Culture of ascitic ovarian cancer cells as a clinically-relevant ex vivo model for the assessment of biological therapies; Metcalf KS et al.; There are few in vitro models of human ovarian cancer suitable for the assessment of biological therapies . We have established short-term cultures of ovarian carcinoma cells from ascites, maintained in suspension in ascitic fluid to help preserve the original tumor cell microenvironment . We assessed the effects of a potential biological therapeutic agent, interferon-alpha (IFN alpha), on ovarian cell phenotype . In total, eight cultures were established from seven patients . Quantitative changes in cell surface phenotype were determined by flow cytometry for HLA-ABC, HLA-DR, TAG72, CA125, HMFG1 and HMFG2 antigens . The amount of CA125 antigen shed into the culture media was also assessed . Variations in cell surface phenotype between specimens probably reflected the specific cytokine milieu of the ascites as well as idiotypic differences between tumors . Nevertheless, there were consistent phenotypic responses to IFN alpha, with up-regulation of MHC Class I but down-regulation of the HMFG1 and HMFG2 antigens from the cell surface . The results suggest that this approach may be useful in patient selection and for optimizing biological therapies, as it enables patients' individual tumor responses to exogenous cytokine to be studied against the background of the endogenous cytokine milieu.

J Lipid Res, 1998 May, 39(5), 987 - 98
Plasma and fibroblasts of Tangier disease patients are disturbed in transferring phospholipids onto apolipoprotein A-I; von Eckardstein A et al.; Plasmas of patients with Tangier disease (TD) lack lipid-rich alpha-HDL which, in normal plasma, constitutes the majority of high density lipoprotein (HDL) . Residual amounts of apolipoprotein (apo)A-I in TD plasma occur as lipid-poor or even lipid-free prebeta-HDL . By contrast to normal plasma, TD plasma does not convert prebeta-HDL into alpha-HDL . Moreover, fibroblasts of TD patients were found to be defective in secreting cholesterol or phospholipids in the presence of lipid-free apoA-I . We have therefore hypothesized that both defective conversion of prebeta-HDL into alpha-HDL and defective lipid efflux from TD cells onto lipid-free apoA-I result from a disturbance in phospholipid transfer occurring in both cellular and extracellular compartments . To test this hypothesis we established an assay that measures the activity of plasma, cells, and cell culture media to transfer radiolabeled phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) from vesicles onto apoA-I, apoA-II, albumin, or reconstituted HDL . Plasmas, HDL, and lipoprotein-depleted plasma of normolipidemic probands as well as cell homogenates and culture media of normal fibroblasts were active at 37 degrees C but not at 4 degrees C in transferring radiolabeled PC, PI, and PE dose- and time-dependently onto either lipid-free apoA-I or reconstituted HDL . Transfer of glycerophospholipids onto apoA-II was much lower than onto apoA-I; transfer onto albumin was close to background . Compared to ten normolipidemic plasmas and four apoA-I-deficient plasmas, plasmas of six TD patients were significantly reduced by 40-50% in their glycerophospholipid transfer activities . Compared to eight normal fibroblast cell lines, homogenates and culture media of four TD fibroblast cell lines were reduced by 40-50% and 30-35%, respectively, in their activity to transfer PC, PI, or PE onto apoA-I . Our data suggest that in TD the same mechanism underlies both defective conversion of prebeta-HDL into alpha-HDL and impaired efflux of cellular lipids, namely a defective phospholipid transfer.

Nephron, 1998, 79(1), 38 - 43
The fibronectin production is increased by thrombospondin via activation of TGF-beta in cultured human mesangial cells; Tada H et al.; Thrombospondin (TSP) is a multifunctional glycoprotein that is synthesized by a variety of cells including mesangial cells (MCs) . To clarify the effect of TSP on the pathogenesis of diabetic nephropathy, we studied the effect of glucose concentrations on TSP synthesis in cultured human MCs . Thereafter, the effects of TSP on the activation of transforming growth factor beta (TGF-beta) and fibronectin production were investigated in MCs . Incubating MCs with elevated glucose levels for 6 days resulted in an increase in TSP synthesis, measured by an enzyme-linked immunosorbent assay, both in culture media and cell layers . Treatment of MCs with TSP (final concentrations 1 and 5 microg/ml) for 24 h resulted in an increase (1.3- and 2.1-fold, respectively) in active TGF-beta, which was determined with an enzyme-linked immunosorbent assay using TGF-beta-soluble receptor type II, in the culture media without having any effect on the production of total TGF-beta . Exposure of MCs to TSP caused enhancement of fibronectin production in both media and cell layers in a TSP dose-dependent manner with the maximum at a TSP concentration of 1 microg/ml . The TSP-induced increase in fibronectin production from MCs was completely prevented by concomitant treatment with 10 microg/ml anti-TGF-beta neutralizing antibody . These results indicate that the TSP production is promoted by a high ambient glucose concentration in human MCs and that TSP, in turn, causes an increase in fibronectin production via activation of TGF-beta.

Virus Genes, 1998, 16(2), 149 - 52
A single amino-acid substitution in gag p19 protein (MA) of Rous sarcoma virus suppresses virus production from infected cells; Hara H et al.; Gag p19 protein (MA) of the transformation-defective Rous sarcoma virus mutant, tdPH2010, has a point mutation at nucleotide 376 (G to A) that results in an amino-acid change at residue 126 of p19 (Glu to Lys) . This single amino-acid change is the cause of the aberrantly fast migration of this protein on SDS-polyacrylamide gels . To study the biological significance of the mutation, we introduced this mutation into a transformation-defective derivative of the molecularly cloned Rous sarcoma virus, SRA2, and examined its effect on virus replication . The virus possessing the mutation in its gag p19 gene had 50% slower replication as measured by the amount of reverse transcriptase as well as gag p27 protein (CA) in the culture media . Glu at position 126 appears to be important for efficient production of Rous sarcoma virus in vitro.

Microbiologia, 1997 Dec, 13(4), 493 - 8
Development of a selective culture medium for Fusarium moniliforme; Castella G et al.; Nash and Snyder medium and malachite green agar 2.5 ppm medium, a new selective culture medium designed in our laboratory, were challenged with pure cultures of Fusarium moniliforme strains and two different mixed-conidium suspensions, which included rapidly spreading fungi, for their utility in the isolation and enumeration of F . moniliforme . From the results of this comparative study, malachite green agar 2.5 ppm allowed only the selective growth of F . moniliforme whereas Nash and Snyder medium allowed both the growth of F . moniliforme and other species not belonging to Fusarium spp . The enumeration of F . moniliforme propagules was similar in both culture media.

J Gastroenterol, 1998 Apr, 33(2), 213 - 7
Increased mRNA expression of a novel prostacyclin-stimulating factor in human colon cancer; Umeda F et al.; We recently cloned a prostacyclin (PGl2)-stimulating factor (PSF), which stimulates PGl2 production by cultured vascular endothelial cells . Immunohistochemistry and Northern blot analysis demonstrated that PSF was highly expressed in colon cancer sites compared with normal colon mucosa obtained from the same patient, as well as in cultured adenocarcinoma cell lines compared with cultured normal colon mucosal cell lines . Increased levels of the PSF protein were detected in the culture media of these adenocarcinoma cells compared with levels in the culture media of normal mucosal cells . These results suggest that PSF is closely associated with carcinogenesis of colon mucosa.

J Environ Sci Health B, 1998 May, 33(3), 253 - 66
Metabolism of carbofuran by Aspergillus niger and Fusarium graminearum; Salama AK; Metabolism of carbofuran in pure liquid cultures of A . niger and F . graminearum was investigated . Carbofuran and its metabolites were analyzed by HPLC and TLC . The average recoveries of carbofuran, 3-hydroxycarbofuran, 3-ketocarbofuran, and 3-ketocarbofuran phenol from fungi media were found to be 88.65, 86.19, 75.48, and 80.48%, while their detection limits were 0.035, 0.031, 0.015 and 0.140 ppm, respectively . The data showed that A . niger was capable of degrading carbofuran more faster than F . graminearum . Carbofuran disappeared biexponentially from the liquid culture media of both fungi . The terminal half-life values of carbofuran were 10.4 and 12 days in the media of A . niger and F . graminearum, respectively . The amounts of carbofuran reached 15.56 and 19.71% of the applied dose after 21 days in case of A . niger and F . graminearum, respectively . Carbofuran was biotransformed to 3-hydroxycarbofuran, 3-ketocarbofuran, and 3-ketocarbofuran phenol . The percentages of the major metabolite, 3-hydroxycarbofuran were 47.72 and 7.77% in case of A . niger and F . graminearum after 21 days, respectively.

In Vitro Cell Dev Biol Anim, 1998 Jan, 34(1), 40 - 5
Primary culture of choroidal epithelial cells: characterization of an in vitro model of blood-CSF barrier; Zheng W et al.; A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier . Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml . The procedure yielded 2-5 x 10(4) cells from pooled plexuses of three to four rats, and a viability of 77-85% . The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2-3 d . The cultures were of distinct choroidal epithelial origins . For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia . Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures . The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers . The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 +/- 10 ohm per cm2) . The epithelial barriers appeared to actively transport {125I}-thyroxine from the basal to apical chamber . These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood-CSF barrier.

Microb Pathog, 1998 Mar, 24(3), 197 - 201
Influence of mycobacterial virulence and culture condition on gamma delta T cell activation; Schoel B et al.; Activation of human gamma delta T cells by culture supernatants of virulent and avirulent mycobacteria was examined . The stimulatory potential of mycobacteria was influenced by the type of culture media and independent from their virulence . Activation of gamma delta T cells by phagocytes infected with viable virulent Mycobacterium tuberculosis H37Rv and avirulent M . bovis BCG was comparable . We conclude that gamma delta T cell stimulation occurs in response to infection with mycobacteria independent from their virulence .

Biol Reprod, 1998 May, 58(5), 1316 - 20
Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development in vitro; Abeydeera LR et al.; In the present study, attempts were made to develop a protein-free (PF) in vitro maturation (IVM) system for pig oocytes and to examine subsequent embryo development after in vitro fertilization . In experiment 1, four IVM media were tested: 1) control: North Carolina State University (NCSU) 23+10% porcine follicular fluid; 2) PF-NCSU: NCSU 23+0.1% polyvinyl alcohol (PVA)+1% amino acids; 3) PF-TCM: Tissue culture medium (TCM) 199+PVA; and 4) PF-WM: PF-Waymouth MB 752/1 medium (WM)+PVA . Oocytes were cultured in the respective media containing eCG and hCG (10 IU/ml each) for 20-22 h and then without hormonal supplements for an additional 20-22 h . After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined . Some oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h in modified Tris-buffered medium containing caffeine and BSA . In experiment 2, oocytes were matured in control, PF-TCM, and PF-WM, fertilized in vitro, and cultured for 144 h in NSCU 23+BSA . Fewer (p < 0.01) oocytes reached metaphase II stage in PF-NCSU (45% vs . 80-85%) than in the other media . Oocytes matured in control medium showed the most cumulus expansion, followed by those in PF-TCM and PF-WM; those in PF-NCSU showed very slight expansion . A lower (p < 0.05) penetration rate was obtained for oocytes matured in PF-NCSU than in the control medium (59% vs . 81%) . In contrast to those in control (96%) and PF-TCM (93%), oocytes in PF-WM (65%) showed a lower male pronuclear formation . Compared to that in the control, a significantly lower (p < 0.05) cleavage rate was also observed for oocytes matured in PF-WM . Similar proportions of embryos developed to the blastocyst stage when oocytes were matured in control (22%) and PF-TCM (13%) . These results indicate that pig oocytes can be successfully matured in a protein-free medium with subsequent development to the blastocyst stage.

FEBS Lett, 1998 Apr 17, 426(2), 233 - 7
Adrenomedullin production is correlated with differentiation in human leukemia cell lines and peripheral blood monocytes; Kubo A et al.; We demonstrated that adrenomedullin (AM) is produced and secreted from human leukemia cell lines (THP-1 and HL-60) as well as peripheral blood granulocytes, lymphocytes, monocytes and monocyte-derived macrophages . Immunoreactive AM accumulated in the culture media of THP-1 and HL-60 cells increased according to their differentiation into macrophage-like cells . Retinoic acid exerted synergistic effects on AM secretion from THP-1 and HL-60 cells when administered with tumor necrosis factor-alpha, lipopolysaccharide or 12-O-tetradecanoyl phorbol-13-acetate . AM was shown to increase the scavenger receptor activity on THP-1 cells . Thus, monocytes/macrophages should be recognized as sources of AM, and the secreted AM may modulate the function of macrophages.

Surg Endosc, 1998 May, 12(5), 436 - 9
An in vitro model fails to demonstrate aerosolization of tumor cells; Sellers GJ et al.; BACKGROUND: We investigated the ability of pressurized CO2 gas to aerosolize B16 melanoma (B16) tumor cells in an in vitro model . METHODS: The experimental apparatus consisted of an 18.9-L plastic cylindrical vessel and a compliant latex pouch was attached to the top . Two 5-mm ports penetrated the vessel; insufflation and desufflation were carried out through them . A culture dish containing 20 million B16 cells in liquid culture media was placed at the base within the container . In the first experiment, the vessel was insufflated with CO2 gas to a static pressure of 15 or 30 mm Hg with the outflow port closed . After 10 min, the outflow port was opened and the gas was desufflated through a collecting device containing sterile culture medium . In a second experiment, a continuous flow of CO2 through the vessel was maintained after a pressure of 15 or 30 mm Hg was established . A total of 10 L CO2 was cycled through the vessel . In both experiments, 24 determinations were carried out at each pressure . Each experimental culture dish was microscopically scanned for 2 weeks for the presence of tumor cells . The third and fourth experiments tested for the presence of aerosolized nonviable tumor cells in the expelled gas . Using the model described above, after 10 mins of 30 mm Hg static pressure, the CO2 gas was expelled directly onto a glass slide and cytofixed . Alternately, after 10 mins at 30 mm Hg static pressure, the gas was expelled through a saline-filled Soluset (Abbott Laboratories), centrifuged, and the residue cytofixed onto a glass slide . Each of the five slides per experiment were examined microscopically for the presence of cells . RESULTS: In the first and second experiments, no cells or growth were observed in any of the 96 experimental dishes . In experiments three and four, no cells were detected on any of the slides . CONCLUSIONS: It was not possible with this model to aerosolize tumor cells in a pressurized CO2 environment . Our results suggest that aerosolization of tumor cells is not the mechanism of port site recurrences after laparoscopic surgery for malignant disease.

J Physiol, 1998 Mar 15, 507 ( Pt 3), 639 - 52
Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors; Krishek BJ et al.; 1 . The interaction of Zn2+ and H+ ions with GABAA receptors was examined using Xenopus laevis oocytes expressing recombinant GABAA receptors composed of subunits selected from alpha1, beta1, gamma2S and delta types, and by using cultured rat cerebellar granule neurones . 2 . The potency of Zn2+ as a non-competitive antagonist of GABA-activated responses on alpha1beta1 receptors was reduced by lowering the external pH from 7.4 to 5.4, increasing the Zn2+ IC50 value from 1.2 to 58.3 microM . Zinc-induced inhibition was largely unaffected by alkaline pH up to pH 9.4 . 3 . For alpha1beta1delta subunits, concentration-response curves for GABA were displaced laterally by Zn2+ in accordance with a novel mixed/competitive-type inhibition . The Zn2+ IC50 at pH 7.4 was 16.3 microM . Acidification of Ringer solution resulted in a reduced antagonism by Zn2+ (IC50, 49.0 microM) without affecting the type of inhibition . At pH 9.4, Zn2+ inhibition remained unaffected . 4 . The addition of the gamma2S subunit to the alpha1beta1delta construct caused a marked reduction in the potency of Zn2+ (IC50, 615 microM), comparable to that observed with alpha1beta1gamma2S receptors (IC50 639 microM) . GABA concentration-response curves were depressed in a mixed/non-competitive fashion . 5 . In cultured cerebellar granule neurones, Zn2+ inhibited responses to GABA in a concentration-dependent manner . Lowering external pH from 7.4 to 6.4 increased the IC50 from 139 to 253 microM . 6 . The type of inhibition exhibited by Zn2+ on cerebellar granule neurones, previously grown in high K+-containing culture media, was complex, with the GABA concentration-response curves shifting laterally with reduced slopes and similar maxima . The Zn2+-induced shift in the GABA EC50 values was reduced by lowering the external pH from 7.4 to 6.4 . 7 . The interaction of H+ and Zn2+ ions on GABAA receptors suggests that they share either a common regulatory pathway or coincident binding sites on the receptor protein . The apparent competitive mode of block induced by Zn2+ on alpha1beta1delta receptors is shared by GABAA receptors on cerebellar granule neurones, which are known to express delta-subunit-containing receptors . This novel mechanism is masked when a gamma2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABAA receptors to endogenous cations.

Fertil Steril, 1998 May, 69(5), 970 - 1
Culturing human embryos with and without glucose; De Silva M; OBJECTIVE: To review published data and to compare pregnancy rates (PRs) after culturing human embryos with and without glucose and phosphate . DESIGN: Comparison of results from various programs . SETTING: Assisted Reproductive Technology Program . PATIENT(S): Patients were enrolled in various studies . INTERVENTION(S): Human embryos were cultured with and without glucose and phosphate . MAIN OUTCOME MEASURE(S): Pregnancy rates after different techniques of embryo culture . RESULT(S): Some studies reported higher PRs in patients undergoing IVF after embryos were cultured in media without glucose and phosphate versus media with glucose and phosphate . One study showed that PRs were lower when embryos were cultured in media lacking glucose and phosphate compared with media containing glucose and phosphate . Some studies have also shown similar PRs with the two types of culture media . CONCLUSION(S): The PRs in IVF patients will not necessarily be enhanced if the embryos are cultured in media without glucose and phosphate.

Neurochem Int, 1998 Mar, 32(3), 265 - 71
Characterization of cyclothiazide-enhanced kainate excitotoxicity in rat hippocampal cultures; Ohno K et al.; Cyclothiazide has been shown to block desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring receptors and to enhance quisqualate-, AMPA- and kainate-induced neurotoxicity . The pharmacology behind this cyclothiazide-enhanced kainate-induced excitotoxicity was characterized in embryonic rat hippocampal cell cultures . Treatment of cell cultures with a combination of cyclothiazide and kainate for 24 h resulted in excessive neuronal death as measured by the release of lactate dehydrogenase into the culture media . Cyclothiazide produced a leftward shift of the kainate dose-response curve and enhanced the maximum response of kainate excitotoxicity . AMPA-preferring receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline(NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) blocked cyclothiazide-enhanced kainate toxicity completely, and cyclothiazide increased the IC50S for NBQX and GYKI 52466 against kainate toxicity . The N-methyl-D-aspartate (NMDA) antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo{a,d} cyclohepten-5,10-imine (MK801) also blocked cyclothiazide-enhanced kainate toxicity, but only partially . Cyclothiazide also increased the IC50 for MK801 against kainate toxicity . These data suggest that cyclothiazide enhances both AMPA-preferring receptor- and NMDA receptor-mediated toxicity in kainate-induced toxicity in embryonic rat hippocampal cultures.

Arch Toxicol, 1998 Mar, 72(4), 197 - 202
Glutathione modifies the toxicity of triethyltin and trimethyltin in C6 glioma cells; Cookson MR et al.; It has been demonstrated that exposure to mercury or cadmium compounds causes alterations in the glutathione system in a model glial cell line, C6 . Here we report that two organic tin compounds, triethyltin (TET) and trimethyltin (TMT), are also toxic to these cells with EC50 values for cell death of c . 0.02 microM and 0.8 microM respectively . Exposure for 24 h to either of these compounds at sub-toxic concentrations caused increases in the amount of reduced glutathione (GSH) per cell . Increases in glutathione-S-transferase enzyme activity were also demonstrated after TET or TMT exposure . This suggests that glutathione increases occur in glial cells after toxic insults below that required to cause cell death, possibly acting as a protective mechanism . To test whether GSH plays a role in organotin-induced cell death we manipulated GSH in the culture media or via intracellular GSH and looked at the effects on sensitivity to TET or TMT toxicity . Adding GSH to the culture media did not protect the cells . Depletion of intracellular GSH with buthionine-{S,R} sulphoximine did not alter cytotoxicity of TET or TMT . However, pre-treatment with (-)-2-oxo-4-thiazolidine carboxylic acid (OTC), which increases intracellular GSH levels, protected the cells against both compounds . The EC50 for TMT was increased from 0.77 to 1.8 microM, a 2.3-fold shift, whereas the EC50 for TET was increased > 20-fold, from 0.022 to 0.47 microM . One interpretation of these results is that GSH protects cells against the toxicity of organic tin compounds without reacting directly with them to any significant extent . Under conditions where GSH is depleted, additional protective mechanisms may be active.

Free Radic Biol Med, 1998 Mar 15, 24(5), 859 - 62
Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors; Schmidt K et al.; We investigated the effects of organic buffers on the NO-like biological activities of ONOO- . In HEPES buffer (50 mM), ONOO- (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO . ONOO- produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM) . Electrochemical detection of NO showed that the biological effects of ONOO- in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions . CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway . Formation of NO from ONOO- was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO- on cGMP accumulation in this buffer . These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO-.

Free Radic Biol Med, 1998 Mar 15, 24(5), 798 - 808
Riboflavin-mediated axonal degeneration of postnatal retinal ganglion cells in vitro is related to the formation of free radicals; Lucius R et al.; It is well known that glial cells produce several neurotrophic factors . We detected a neurogedegenerative/neurite growth inhibiting activity in serum-free astrocyte-conditioned medium (ACM) . After high performance liquid chromatography (HPLC)-purification, spectral analysis and test of biologic activity in tissue cultures of postnatal retinal explants we isolated a fraction containing a riboflavin-(vitamin B2)-like compound which caused the neuronal degeneration . We therefore investigated the influence of pure riboflavin on axonal regeneration in vitro . Riboflavin is a normal compound of Dulbecco's modified Eagle medium (DMEM) and other tissue culture media in various concentrations . The removal of riboflavin from ACM by reversed phase chromatography abolished the neurite growth inhibiting effect and enhanced the regenerative response of axonal outgrowth from postnatal rat retinal explants . However, doubling of the normal medium concentration (1 microM) of riboflavin lead to strong degenerative alteration of the outgrowing axons in a dose-dependent manner, even under maximal growth stimulation by cultivating the explants in astrocyte-conditioned medium . To check the possibility that riboflavin-mediated cytotoxicity is related to the production of free radicals through photoabsorption from daylight, we irradiated culture medium with UV light, and induced radical stress by incubating the explants with Fe2+/3+ . In an other set of experiments, we proofed, if antioxidants/free radical scavengers like pyruvate or vitamin C and E are able to overcome the neurite growth inhibiting influence of riboflavin or the radical stress . Our findings suggest an involvement of riboflavin-mediated formation of free radicals/reactive oxygen species and subsequent neurite degeneration in in vitro-assays of neuronal regeneration or neuronal cell cultures . How far the riboflavin/free radical-induced axonal degeneration could be an explanation for neurological degenerative disorders has to be elucidated.

Environ Mol Mutagen, 1998, 31(3), 218 - 27
Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1,650 subjects in an Italian population: I . Contribution of methodological factors; Barale R et al.; The influence of several methodological factors on mean values of sister chromatid exchanges (SCEs) and micronuclei (MN) in peripheral lymphocytes of 1,650 subjects was analyzed . Donors belonged to a general healthy population living in Pisa and in two nearby small cities: Cascina and Navacchio (Ca-Na) . Blood samples were collected over a period of 29 months and processed in three different laboratories of the some institute . Slides were analyzed by several scorers . Our data showed that lymphocyte proliferation indexes (PIs) and baseline mean values of SCEs were affected mainly by sampling period . This factor accounted for a percentage ranging from roughly 10% (Pisa) to 20% (Ca-Na) of total SCE variance and from roughly 10% (Pisa) to 13% (Ca-Na) of total PIs variance . A marginal effect was attributable to the different laboratories involved (maximum 3% for SCEs and 7% for PIs) . The sampling period variable included many sources of variability such as culture media batches, fetal calf serum, PHA, BrdUrd, and seasonality . MN counts revealed a more marked dependence on processing laboratories . This factor accounted for a percentage of roughly 10% (Pisa and Ca-Na) of total variance, while the sampling period was marginally effective (about 1-4% of total variability) . Because laboratories were equipped and supplied with the same materials and consumables and technicians were rotated constantly, the only variable ascertained was represented by the three different models of CO2 incubators used for lymphocyte culturing . When "month" and "incubator" variables were considered jointly, experimental variability accounted for 15-20% of total variance, both for PIs and mean values SCEs and MN . The variability due to slide scoring was reduced by assigning each slide to five different scorers and matching low with high scorers in each group . Present data show that when the study is performed under these controlled conditions, about 20% of total interdonor variability can be explained by experimental or seasonal factors.

Hum Reprod Update, 1997 Nov-Dec, 3(6), 541 - 52
The baboon oviduct: characteristics of an oestradiol-dependent oviduct-specific glycoprotein; Verhage HG et al.; The baboon oviductal epithelium differentiates into a tall columnar epithelium consisting of ciliated and secretory cells during the follicular phase of the menstrual cycle in response to rising oestradiol levels . The apical tips of these secretory cells are filled with membrane-bound secretory granules . During the luteal phase when progesterone levels are elevated, the epithelium regresses and deciliation occurs . Analysis of secretory proteins obtained from explant culture media by SDS-PAGE followed by fluorography or Western blots has revealed that the baboon oviduct synthesizes and secretes a high molecular weight glycoprotein during the follicular phase of the cycle . Immunocytochemistry demonstrated that this oviductal glycoprotein is localized to the secretory granules of epithelial secretory cells, is oviduct specific, and that following secretion the oviductal glycoprotein binds to the zona pellucida and perivitelline space of ovulated oocytes and embryos within the oviduct . Similar proteins have been characterized in other mammalian species . cDNA data show that the complete coding sequence is 2228 bp for a protein of 623 amino acids . A Genbank search showed that baboon oviductal glycoprotein has high homology to other oviductal glycoprotein sequences at both the nucleotide and amino acid levels . Studies conducted to date probing the biological function of oviductal glycoprotein indicate that this protein plays a role in prefertilization reproductive events (sperm capacitation; sperm-zona binding; zona penetration) . Additional experiments are needed to reveal a specific function and mechanism for this molecule.

Invest Ophthalmol Vis Sci, 1998 May, 39(6), 922 - 36
Area and depth of surfactant-induced corneal injury correlates with cell death; Jester JV et al.; PURPOSE: In previous studies in which in vivo confocal microscopy (CM) was used, quantifiable differences were identified in the corneal epithelium and stroma for surfactants producing different degrees of ocular irritation . In the present study, in vivo confocal microscopy was used to determine area and depth of the initial corneal changes, and the correlation of the data to cell death was characterized by ex vivo live-dead assay . METHODS: In four groups of rabbits (12 animals each), 10 microl surfactants known to produce slight, mild, moderate, or severe irritation was applied to the central cornea of one eye; 4 untreated rabbits served as controls . Measurements of group total mean epithelial thickness, epithelial cell area, and depth of keratocyte loss in four corneal regions were made by in vivo CM in 6 rabbits of each group and in 4 control animals at 3 hours and in the remaining rabbits at 3 hours and 1 day . Corneas were then removed and fixed for conventional histologic examination (two eyes/treatment/group), or regions were excised and placed in culture media containing 2 microM calcein-acetoxymethyl ester (calcein-AM) and 4 microM ethidium homodimer . Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 microm (in the x, y, and z axes, respectively) volume of the cornea was determined . RESULTS: Confocal microscopy showed that application of the slight irritant resulted in decreased epithelial thickness at 3 hours (41.2+/-2.6 microm in treated eyes versus 43.6+/-3 microm in control eyes; n=6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630+/-203 microm2 versus 1427.2+/-90.7 microm2) . On day 1, mild, moderate, and severe irritants caused complete loss of epithelium and disappearance of keratocytes to a depth of 30.8+/-10.7 microm, 47.2+/-10.4 microm, and 764.6+/-159.6 microm (n=6, 5, and 6), respectively . At 3 hours, live-dead assay detected more dead epithelial cells as a percentage of total surface cells (49.2+/-4.5% in slightly irritated eyes versus 20.9+/-3.2% in control eyes), significantly correlating with the measurement by in vivo CM of average epithelial cell size in each eye (r=-0.96; P < 0.005) . On day 1, mild and moderate irritants showed increasing stromal cell death from 9.8+/-16.2 cells to 36.4+/-17.7 cells, which significantly correlated with the depth of stromal injury determined by in vivo CM (r=0.79; P < 0.00001) . No surviving keratocytes were detected in severely irritated eyes . CONCLUSIONS: The data support the hypothesis that differences in surfactant-induced ocular irritation are directly related to area and depth of acute corneal injury.

Ophthalmologe, 1998 Mar, 95(3), 148 - 52
{Serum-free cultivation of bovine stromal fibroblasts}; Denk PO et al.; The purpose of the present study was to conduct a comparative evaluation of the effect of several serum-free culture conditions on adhesion, population doubling, cryopreservation and PDGF-induced effects on cell proliferation of bovine stromal fibroblasts (BSF) . Additionally, these effects were compared to serum-containing cultures . METHODS: Only second-passage BSF were used . Cells were cultured using four different culture media (WM/F12, WM/F12 + FCS 1%, LR-1, DMEM) . After 24 h, plating efficiency was determined using a cell-counter system . Subsequently, the cells were seeded at a density of 100 cells/mm2 and cultured for 10 days using the different culture media . Cell number was determined at day 2, 4, 7 and 10 after seeding . Furthermore, the effect of 50 ng/ml PDGF-BB on the proliferation of BSF was tested for these conditions . Cell vitality was determined after cryopreservation of two weeks for each culture medium . RESULTS: The plating efficiency of BSF ranged from 50.2 to 55.5% for the serum-free culture media in contrast to serum-containing conditions, where plating efficiency was 94.8% . With WM/F12 + FCS 1%, a population doubling of 1.27 was observed after an incubation period of 10 days . In contrast, cultivation under serum-free conditions caused neither significant cell proliferation nor cell loss . The stimulation of cell proliferation with PDGF-BB was shown to be 28% (LR1), 40% (WM/F12 + FCS 1%) 76% (WM/F12) and 95% (DMEM) compared to the control . While cell vitality after cryo-preservation was found to be 62.7% using WM/F12 + FCS 1%, vitality using serum-free media was 12.6-22.8% . CONCLUSIONS: The results of the present study demonstrate that with respect to optimal cell adhesion and cell vitality after cryo-preservation, serum-containing media should be used . BSF cultured under the serum-free conditions used in the present study can be maintained quiescent and vital for at least 10 days . Therefore, these serum-free media are useful for cell-culture studies (e.g., determination of proliferation and cytotoxicity).

Eur J Biochem, 1998 Apr 1, 253(1), 67 - 75
Activation of the precursor of human stromelysin 2 and its interactions with other matrix metalloproteinases; Nakamura H et al.; Matrix metalloproteinases (MMP) are synthesized as inactive zymogens (proMMP) and subsequently activated by many factors to degrade the extracellular matrix (ECM) . In the present study, we have examined the intermolecular activation mechanisms of proMMP by MMP-10 (stromelysin 2) . ProMMP-10 was purified from the culture media of OSC-20 human oral squamous carcinoma cells stimulated with 12-O-tetradecanoylphorbol 13-acetate . The final products are partially activated (approximately 38% of the full activity) during the purification steps and contain proMMP-10 of Mr 56,000 with minor protein bands of Mr 47,000, 24,000 and 22,000 . The zymogen is activated by 4-aminophenylmercuric acetate and processed to the active forms of Mr 47,000 and 24,000 . The NH2-terminal sequence of the 47,000- and 24,000-Mr species is Phe82-Ser-Ser-Phe-Pro-Gly, which is identical to that of stromelysin 2 . ProMMP-9 (progelatinase B) is activated by MMP-10 to its full activity and processed to the low-Mr species of Mr 81,000, 65,000, 57,000 and 55,000, the former two of which show proteolytic activity on a gelatin zymography . The NH2-terminal sequence analysis indicates that the 81,000-, 65,000- and 57,000-M, species have the identical sequence of Phe88-Gln-Thr-Phe-Glu-Gly, suggesting the cleavage of the Arg87-Phe88 peptide bond for activation and both NH2-terminal and COOH-terminal truncation in the 65,000- and 57,000-Mr forms . MMP-10 also activates proMMP-7 (promatrilysin) up to about 60% of the full activity and generates the same active species of Mr 19,000 as that obtained by activation with 4-aminophenylmercuric acetate . Incubation of proMMP-2 (progelatinase A) or proMMP-3 with MMP-10 does not result in activation of these proMMP . These results indicate that in addition to the previously reported activation of proMMP-1 (tissue procollagenase) and proMMP-8 (neutrophil procollagenase), MMP-10 can also activate proMMP-9 and proMMP-7, and suggest the possibility that MMP-10 may replace a role of MMP-3 in the ECM degradation in concert with other MMP under various pathological conditions.

Am J Physiol, 1998 Apr, 274(4 Pt 1), L508 - 16
H2O2 causes endothelial barrier dysfunction without disrupting the arginine-nitric oxide pathway; Gupta MP et al.; We have previously demonstrated that nitric oxide (.NO) donors attenuate and that inhibition of endogenous nitric oxide synthase (NOS) enhances hydrogen peroxide (H2O2)-mediated porcine pulmonary artery endothelial cell (PAEC) injury . The current study investigates the hypothesis that oxidant-mediated inhibition of NOS contributes to PAEC injury . PAEC barrier function, measured as the transmonolayer clearance of albumin, was significantly impaired by H2O2 (10-100 microM) in the absence of cytotoxicity . Treatment with H2O2 did not alter NOS activity, measured as the conversion of {3H}arginine to {3H}citrulline in PAEC lysates, either immediately after treatment with 0-250 microM H2O2 for 30 min or for up to 120 min after treatment with 100 microM H2O2 . H2O2 had little effect on NOS activity in intact PAECs, measured as 1) the formation of {3H}citrulline in {3H}arginine-loaded PAECs, 2) PAEC guanosine 3',5'-cyclic monophosphate content, and 3) PAEC.NO release to the culture media . These results indicate that the arginine-.NO pathway remains intact after exposure to oxidant conditions sufficient to promote functional derangements of vascular endothelial cells.

Infect Immun, 1998 May, 66(5), 1898 - 903
Identification of the aspartate-beta-semialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant; Harb OS et al.; The ability of Legionella pneumophila to cause Legionnaires' disease is dependent on its capacity to survive in the intracellular environment of its host cells . Furthermore, outbreaks of this disease have been associated with contaminated water sources where L . pneumophila survives as a parasite of protozoa . In this study, we determined the effect of nutritional auxotrophy on the ability of L . pneumophila to survive in the intracellular environment of its host cells . We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L . pneumophila by disruption of the aspartate-beta-semialdehyde (asd) gene . The ability of AA400 to survive within macrophages and protozoa was found to be defective . This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival . Furthermore, the defect was not completely complemented by DAP supplementation to the culture media . Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires' disease.

Graefes Arch Clin Exp Ophthalmol, 1998 Mar, 236(3), 230 - 3
Photosensitization of retinal pigment epithelium by protoporphyrin IX; Bynoe LA et al.; BACKGROUND: Clinical evidence of injury to the retinal pigment epithelium is an important feature of age-related macular degeneration, but the mechanism of this injury is unknown . Blue-light-dependent activation of the blood-borne photosensitizer protoporphyrin IX is known to produce free radicals which may damage cells and tissues . This study was undertaken to determine the effect of blue light and protoporphyrin IX on retinal pigment epithelial cells in vitro . METHODS: Third-passage porcine retinal pigment epithelial cells were plated in six-well culture plates at 100,000 cells/well and grown to confluence . Retinal pigment epithelial cells were then incubated in culture media with and without 35 micrograms/dl protoporphyrin IX and exposed to low intensity (118 microW/cm2) blue, blue-free, or full-spectrum white light in an irradiating incubator for 16 h on/8 h off cycles for 7 days . Some of the wells were shielded from light (dark controls) . Retinal pigment epithelial cells were examined by light microscopy and were trypsinized and counted after 7 days . RESULTS: White light with and without protoporphyrin IX and protoporphyrin IX in dark conditions did not decrease the retinal pigment epithelial cell count significantly . Blue light alone and blue light with protoporphyrin IX decreased the cell count by 22 +/- 4% and 35 +/- 3% compared to the controls, respectively . CONCLUSION: Blue wavelength light without exogenous protoporphyrin IX has a cytotoxic effect on confluent cultures of retinal pigment epithelium, suggesting that endogenous photosensitizers may be present in retinal pigment epithelial cells . Protoporphyrin IX has an additive cytotoxic effect in the presence of blue light, suggesting that this photosensitizer is capable of mediating blue-light-induced retinal pigment epithelial damage . Since protoporphyrin IX is present in blood and tissue fluids, and the retina is chronically exposed to light, protoporphyrin IX-mediated free radical formation may occur in vivo and may play a role in retinal pigment epithelial changes that occur early in the pathogenesis of age-related macular degeneration.

J Steroid Biochem Mol Biol, 1998 Jan, 64(1-2), 1 - 12
Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system; Wei P et al.; The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes . To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR . The intracellular concentration of rGR in E8.2/GR3 cells--from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells--could be manipulated by varying the Tc concentration in the culture media . Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc . Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation . RU486 inhibited both DEX-dependent transactivation and transrepression . DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner . DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc . These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.

What Is Activated Sludge?, What Is Listeria Monocytogenes?, What Is Environmental Microbiology?, What Is Bioengineering?, What Is Biotechnology?, c, Microbe, c, Bacteriology, e, Bacterium, s, Microbiology, r, Microorganism, c, Escherichia coli, n, Salmonella typhimurium, n, Functional genomics, s, Growth media, i, Eubacterium, a, Growth media, s, Clostridia, e, Vibriosis, i, Antibiotics, o, Pseudomonas, n, Multidrug resistant, a, Streptococcal, r, Phage, c, Yeasts, e, Eutrophicated, n, Escherichia coli, e, Ps. putida, a, Pasteurella, c, Streptococci, e, Escherichia coli, a, Sepsis

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

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

Last modified: May 25, 2005