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