Int J Parasitol, 1995 Jan, 25(1), 127 - 30 Echinococcus multilocularis protoscoleces and hepatic cell activity in vitro; Gabrion C et al.; E . multilocularis protoscoleces were co-cultured with hepatic cells in the presence of IAR 20 or BALB/c 3T3 cells . Hepatocyte activity was determined by assaying transferrin and albumin secretion in culture media . The level of these 2 plasma proteins is higher in hepatic/BALB/c 3T3 co-culture medium . In the presence of parasites, the transferrin level is unchanged while the production of albumin is stimulated during the first 48 h . Our results suggest that the albumin production could be attributed to a complex cellular cooperation between hepatocytes and activated Kupffer cells as previously observed in the acute inflammatory reaction. Eur Cytokine Netw, 1995 Jan-Feb, 6(1), 45 - 8 Endogenous nitric oxide production by human monocytic cells regulates LPS-induced TNF production; Zinetti M et al.; The ability to produce nitric oxide (NO) of human monocytes macrophages is object of debate . While studying the regulation of tumor necrosis factor (TNF) synthesis induced by endotoxin (LPS) in a human cell line of monocyte origin (THP-1) and in human peripheral blood mononuclear cells (PBMC) we found an indirect evidence of such production . We showed that L-N-monomethyl-arginine (L-NMMA), an inhibitor of NO synthase, and hemoglobin, a chelator of NO, are able to significantly reduce TNF synthesis, indicating that NO production is induced by LPS and contributes to the induction of TNF . Since NO is a known cytostatic agent, we also studied the cytostatic effect of LPS, and demonstrated that it is reverted by L-NMMA . Although we were unable to show any nitrites/nitrates accumulation in the culture media, taken together our data give an indirect evidence of a physiologically relevant LPS-induced NO production in human monocytes-macrophages. Rev Latinoam Microbiol, 1995 Jan-Mar, 37(1), 71 - 7 {Trypanosoma cruzi: influence of human plasma on the morphogenesis of blood trypomastigotes in a cell-free culture media}; Zaidenberg A et al.; Morphogenesis of blood stream trypomastigotes in the cell free culture medium F69 at 37 degrees C for 10 days showed qualitative differences either with or without human plasma . Without human plasma, blood stream trypomastigotes performed only one cycle before disappearing and the culture kept growing as amastigotes and epimastigotes until the end of the experiment . In contrast, human plasma induced multiple cycles of transformation . The sequence was blood stream trypomastigotes, regressive parasites, amastigotes, progressive parasite and again trypomastigotes . Human plasma preserved the trypomastigote stage, produced a blockade of the epimastigote stage and inhibited the division of amastigotes . In this experimental model, human plasma modified the biological cycle of T . cruzi by inducing or inhibiting different stages. Biol Trace Elem Res, 1995 Jan-Mar, 47(1-3), 57 - 67 Effect of aluminum and lead salts on lipid peroxidation and cell survival in human skin fibroblasts; Dominguez MC et al.; The aim of this study was to see whether aluminum (Al) and lead (Pb) salts are toxic for cultured human fibroblasts under different experimental conditions, in the controllable situation offered by cell cultures . Cell survival and membrane lipid peroxidation served as markers of Al and Pb toxicity . Evaluation of the living cells was carried out using a colorimetric method, the mitochondrial reduction of 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) . Lipoperoxidation assay was performed on whole cell homogenates by measuring thiobarbituric acid-reactive substances (TBARS) produced after incubation with ascorbic acid-ferrous sulfate . Al(III) and Pb(II) salts (300 microM) produce a considerable decrease in cell survival after an exposure period of 4d, evident with the three fetal calf serum concentrations in the culture media: 2, 5, and 10% . Taking into account in vitro cell aging, the cytotoxic effects of Al(III) and Pb(II) are greater in senescent fibroblasts than in young cells . Lead-induced cytotoxicity is higher than Al-induced cytotoxicity . A mechanism that contributes to cellular toxicity is membrane lipid peroxidation; our results demonstrate that Al(III) and Pb(II) ions, 400 microM, exert an antioxidant-like effect or a pro-oxidant action on cell membranes depending on exposure time . We describe significant increases in TBARS formation associated with the presence of 400 microM Al(III) or Pb(II) salts in the culture media . Our study also revealed that these heavy metals induce a cell age-dependent action on membrane lipoperoxidation that is greater in senescent fibroblasts and this could have severe consequences for maintenance of cellular integrity. Biol Trace Elem Res, 1995 Jan-Mar, 47(1-3), 185 - 92 Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures . Relationship with lipid peroxidation; Sergent O et al.; Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM . Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content) . In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes . The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure . An early increase of intracellular LMW iron (< or = 1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas alpha-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron . Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides . Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected . During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation. Hum Reprod, 1995 Jan, 10(1), 171 - 6 Detection of cytokines (interleukin-1, interleukin-6, transforming growth factor-beta) and soluble tumour necrosis factor receptors in embryo culture fluids during in-vitro fertilization; Austgulen R et al.; Several lines of evidence suggest that a number of immunoactive cytokines participate in early reproductive events such as implantation and placental development . Furthermore, cytokines may influence embryo growth and differentiation . In the present study, the production of tumour necrosis factor (TNF), interleukin-1 (IL1), interleukin-6 (IL6) and transforming growth factor-beta (TGF beta) during the first 48 h after oocyte retrieval during in-vitro fertilization was investigated . In addition, the question was raised whether soluble receptors may contribute to cytokine activity regulation in early reproduction, and concentrations of TNF and IL6 receptors in culture media were determined . Finally, an investigation of whether any association exists between cytokine concentrations and embryo morphology was performed . Media from 256 embryos were analysed . IL1, IL6 and TGF beta were produced during the 48 h culture period, whereas no TNF was detected . Levels of IL1 and IL6 were significantly higher in media from the first 24 h culture period than from the second period, whereas TGF beta concentrations in supernatants from the two observation periods did not differ . IL6 receptors were not detected, whereas TNF receptors (p75) appeared in media from the 24-48 h culture period . Granulosa, cumulus and sperm cells are potential sources of cytokine production, especially during the first 24 h period . The contribution of the embryo to cytokine/cytokine receptor production remains an open question . No significant correlation was observed between cytokine/cytokine receptor concentrations and embryo morphological score. Bone, 1995 Jan, 16(1), 171 - 7 Osteoclast formation from human cord blood mononuclear cells co-cultured with mice embryonic metatarsals in the presence of M-CSF; Mbalaviele G et al.; Investigating the potentiality of cord monocytes to differentiate toward osteoclast-like cells (OCL) in vitro, we previously reported that in the presence of 1,25(OH)2 vitamin D3 (1,25-(OH)2D3), multinucleated-cells generated by cord monocyte cultures though displaying morphological features of OCL failed to resorb devitalized bones . We thus hypothesized that full differentiation of cord monocytes toward bone-resorbing cells may require the presence of factors released from and/or direct interactions with living osteogenic cells . In the present study, we tested these hypotheses using two culture systems supporting the development of bone-resorbing cells in the presence of bone matrix . First, cord mononuclear cells were co-cultured with murine fetal metatarsals depleted of osteoclast progenitor cells (stripped metatarsals) in the presence of 1,25-(OH)2D3 . We found that cord mononuclear cells failed to differentiate toward OCL as indicated by the absence of the release of 45Ca previously incorporated in fetal bones and by the absence of formation of TRAP-positive (TRAP{+}) multinucleated cells which have invaded mineralized cartilage during the co-culture period . In the same model, we then investigated the effect of some soluble factors known as stimulators of osteoclast differentiation . Whereas exogenous rhIL6 and rhIL3 were ineffective in this assay, rhM-CSF consistently increased both the number of TRAP(+) multinucleated cells inside the mineralized cartilage and the release of 45Ca into the culture media . The effects of rhM-CSF were time-dependent reaching the maximum after 3 weeks of culture.(ABSTRACT TRUNCATED AT 250 WORDS) J Invest Surg, 1995 Jan-Feb, 8(1), 31 - 42 Inflammatory intermediates produced by tissues encasing silicone breast prostheses; Mena EA et al.; Silicone prostheses, when implanted within the soft tissues of the breast, evoke an inflammatory reaction . In response to silicone exposure, inflammatory mediator production by individual cells has been observed in various experimental studies . In this study, inflammatory mediator production by periprosthetic tissues (whole organ) was measured . The mediator levels were correlated with both the tissue histopathology of the periprosthetic capsules and the clinical symptoms noted by each patient . Tissue surrounding breast implants removed at surgery from ten women (average age and implant duration 40 and 7 years respectively) was cultured in vitro for 24 hours . Control tissues consisting of (a) augmentation mammaplasty skin scars from eight additional patients and (b) knee synovium from seven orthopedic surgery patients with arthritis undergoing primary joint arthroplasty were similarly cultured . The mediators {interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and prostaglandin E2 (PGE2)} liberated into the culture media were measured by an enzyme linked immunosorbent assay . When compared to controls, the mediator levels of IL-6 and TNF-alpha were substantially greater, although IL-2 and PGE2 were lower . Levels varied greatly from patient to patient: in pg/ml per 10 g tissue, IL-2 ranged from 10 to over 1,000; TNF-alpha from 100 to 1,000; IL-6 from 100 to 1,000,000; and PGE2 from 100 to 10,000 . The correlation between TNF-alpha and PGE2 levels was .5 between IL-6 and PGE2 was .6, and between IL-6 and TNF-alpha was .77 . The correlation between TNF-alpha and IL-6 was statistically significant at a p-value less than .01 . Elevated levels of TNF-alpha production were associated with an increased number of macrophages and overall tissue cellularity (p < .05) . No significant relationship was observed between mediator production and clinical symptoms . We conclude that overall cellularity, specifically macrophages, in the periprosthetic capsule may lead to TNF-alpha production but that cytokine production by periprosthetic tissues alone is not a predictor of clinical symptomatology in patients with silicone breast prostheses. Biomaterials, 1995 Jan, 16(1), 51 - 9 Neutrophil-mediated degradation of segmented polyurethanes; Labow RS et al.; The biostability of polyurethanes was evaluated using a human neutrophil cell culture . The polymers were synthesized with 14C radiolabelled components incorporated into the polyurethane chain and the amount of radiolabel released during exposure to cells and medium was used as a marker for material degradation . The effect of diisocyanate, soft segment and chain extender chemistry on the susceptibility of polymer degradation was examined . All polymers showed a release of material into the tissue culture medium which was unrelated to the cells . A significant cell-dependent release of radiolabel-containing material was found from one of the polymers (a polyester urea-urethane, TDI/PCL/ED) which increased linearly up to 96 h . The polyether-containing polyurethanes showed no significant cell-mediated degradation under similar conditions as measured by radiolabel release . Scanning electron microscopy (SEM) showed that the cells adhered to the different polyurethanes . However, no effect of neutrophils on polymer structure could be detected by this technique . The cellular response to each polymer was evaluated by measuring release of elastase-like activity (ELA) into the tissue culture media . After 24h TDI/PCL/ED showed the highest levels of ELA in the tissue culture medium . When TDI/PCL/ED was incubated with commercial elastase in vitro, a significant release of radiolabel was found which was comparable to the amount of radiolabelled material released from this polymer in contact with the neutrophils in culture . No significant amount of radiolabel was released from the corresponding polyether material (TDI/PTMO/ED) under similar conditions.(ABSTRACT TRUNCATED AT 250 WORDS) Insect Biochem Mol Biol, 1995 Jan, 25(1), 119 - 26 Characterization of a spectrophotometric assay for juvenile hormone esterase; McCutchen BF et al.; Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify juvenile hormone esterase (JHE) activity . The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs . As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol . In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm . Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-1-hexylthioacetate, were compared for JHE activity . Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester . However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined . Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays . Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate . Several esterase bands were found at different isoelectric points when gels were stained with alpha-naphthyl acetate.(ABSTRACT TRUNCATED AT 250 WORDS) J Reprod Fertil, 1995 Jan, 103(1), 33 - 9 Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival; Mahmoudzadeh AR et al.; Experiments were designed to determine optimal conditions for the cryopreservation of bovine embryos produced in vitro . In Expt 1, embryos were exposed for 1, 3 or 5 min to a vitrification solution consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll and 10.26% (w/v) sucrose (EFS) and were subsequently vitrified . After warming in water at room temperature and diluting in a solution of 0.25 mol sucrose l-1, the in vitro survival rate in Menezo-B2 medium was highest after exposure to EFS for 1 min . In Expt 2, embryos at day 7 and day 8 were vitrified after exposure to EFS for 1 min . The survival rate of embryos at day 7 was significantly improved, especially at the blastocyst and expanded blastocyst stage, when the Menezo-B2 medium was supplemented with bovine oviduct epithelial cells (BOEC) . Embryos at day 8 exhibited a significantly lower survival rate than did embryos at day 7 in both culture media . In Expt 3, one-step exposure of embryos to EFS for 1 min was compared with two-step exposure to 20% ethylene glycol for 3 min and EFS for 30-45 s . Embryos exhibited significantly higher survival and hatching rates after two-step vitrification, especially at the expanded blastocyst (89% and 69%, respectively) and the blastocyst stage (75% and 38%, respectively) . In Expt 4, embryos were diluted in solutions of 0, 0.25 or 0.5 mol sucrose l-1 after two-step vitrification . There were no significant differences in the survival rates between the three dilution treatments.(ABSTRACT TRUNCATED AT 250 WORDS) J Appl Biomater, 1995 Spring, 6(1), 65 - 8 Fibroblast seeding and culture in biodegradable porous substrates; Rivard CH et al.; A natural poly(hydroxybutyrate-co-9% hydroxyvalerate) copolyester was processed into a three-dimensional porous foam structure by salt leaching/solvent casting with previously sieved sodium chloride salts . Laboratory-built P(HB-9% HV) foams and commercial collagen sponges were cut into small rectangular specimens, sterilized, and prewetted using ethanol, rinsed with Dulbecco's minimum essential medium + 10% serum culture media, and seeded with fibroblasts isolated from canine anterior cruciate ligaments . The fibroblast cultures into such porous substrates were performed from 0 to 35 days by incubation (5% CO2) at 37 degrees C . It demonstrated that the P(HB-HV) sustained a cell proliferation rate similar to that observed in collagen sponges, up to at least 35 days, with a maximal cell density on the day 28 in culture . On the other hand, the P(HB-HV) materials kept their structural integrity during the culture period while the collagen foams contracted greatly . Further, the total protein production after 4 weeks in culture was found to be twice as high (190 +/- 10%) in the P(HB-9% HV) foam than in the collagen foam . Porous P(HB-HV) materials appear to be adequate polymeric substrates for cell cultures . However, further evaluations are still required to confirm such preliminary results. Dev Dyn, 1995 Jan, 202(1), 91 - 9 Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells; Guerin CW et al.; The expression of matrix-degrading metalloproteases (MMPs) by human skeletal muscle satellite cells was investigated by zymography of cell culture media and by Northern blot analysis of mRNA prepared from satellite cells . Zymography in gelatin substrate gels revealed that satellite cells constitutively synthesize and secrete 72 kDa gelatinase (MMP-2) . In addition, treatment of satellite cell cultures with phorbol ester resulted in an induction of 92 kDa gelatinase (MMP-9) activity . On casein substrate gels, little or no proteolytic activity was detectable in control or phorbol ester treated satellite cell cultures, suggesting that compared to fibroblasts, satellite cells secrete little or no interstitial collagenase (MMP-1) or stromelysin (MMP-3) activity . Northern blotting, however, revealed that there is detectable expression of mRNA transcripts encoding MMP-1 in satellite cell cultures, and that increased accumulation of MMP-1 mRNA transcripts occurs upon treatment of these cells with phorbol ester . In contrast, no constitutive, or induced expression of transcripts encoding MMP-3 was detectable in satellite cells . These findings show that satellite cells can synthesize and secrete selected members of the MMP family and suggest that skeletal muscle cells may participate directly in remodelling of the extracellular matrix during myogenesis and the regeneration of skeletal muscle. Vestn Ross Akad Med Nauk, 1995, (7), 28 - 9 {Determination of drug sensitivity of Mycobacterium tuberculosis on solid and liquid culture media}; Korneev AA et al.; The paper gives a comparative characterization of some unfavourable factors influencing antituberculous agents which are a part of dense egg and liquid media in order to determine the drug resistance of Mycobacterium tuberculosis . The liquid media are more preferable primarily, mainly by preserving the activity of antituberculous agents to set accelerated methods for the detection of the spectrum of and the resistance of clinical mycobacterial isolates. Res Exp Med (Berl), 1995, 195(2), 101 - 16 Photodynamic therapy decreases cancer colonic cell adhesiveness and metastatic potential; Vonarx V et al.; Plasma membrane damage induced in various cell targets by hematoporphyrin (HPD) photodynamic therapy (PDT) could modify cancer cell adhesiveness, an important parameter in cancer metastasis . We investigated the effect of HPD or HPD incubation followed by argon laser light on the adhesiveness of progressive (PROb) or regressive (REGb) cancer cells of the same colonic origin but with a different in vivo metastatic potential . Adhesiveness was studied on plastic or endothelial cell monolayers (ECM) . In the absence of treatment, both PROb and REGb cells adhered better on plastic than on ECM . HPD alone or HPD-PDT induced toxicity proportional to the HPD dose . HPD-PDT increased the adhesiveness rate of both cell lines on plastic and decreased it on ECM . HPD-PDT of ECM increased adhesiveness, but only at HPD doses causing at least 50% cell death . With HPD treatment alone or HPD-PDT of culture media, there was no significant decrease in cell adhesiveness to ECM . We also studied the effect of HPD or HPD incubation followed by argon laser light on the metastatic potential of cancer cells, which was decreased for PROb with HPD alone or HPD-PDT . Decreased adhesiveness of colonic cancer cells to ECM after HPD-PDT was thus correlated with decreased metastatic potential . REGb cells did not acquire a progressive phenotype either in vitro or in vivo after HPD-PDT. Mycoses, 1995, 38 Suppl 1, 55 - 63 {Comparison of seven methods of in vitro susceptibility testing of clinical yeast isolates against fluconazole}; Schmalreck AF et al.; Four commercially available in vitro test systems (Candifast, E-test, Mycototal, Spiral-Gradient Endpoint Method), agardiffusion with 25 micrograms fluconazole paper test discs and 15 micrograms test tablets, and agardilution were compared to the microbroth dilution method by fluconazole susceptibility testing of 145 clinical isolates . In addition, the culture media provided or recommended by the manufacturers of the test systems were compared to the high resolution (HR) antifungal test medium . With all currently available culture media growth problems (inhibition or delayed growth of the clinical isolates) occurred with solid or semi-solid media . With minor improvements, HR medium demonstrated the most reproducible and comparable results (supplementation with asparagine and deletion of sodium hydrogen carbonate) . Best correlation to microdilution was obtained by the agardilution method > (95% concordance) followed by the spiral gradient endpoint method (85%), Candifast (83%), Mycototal (81%) and the E-test (78%) . Regression analysis demonstrated good correlation between agardiffusion and micro-/agardilution(r > 0.9). Adv Enzyme Regul, 1995, 35, 91 - 100 Glucuronidation by human colorectal adenocarcinoma cells as a mechanism of resistance to mycophenolic acid; Franklin TJ et al.; Mycophenolic acid (MPA), a potent and specific inhibitor of IMP dehydrogenase, exerts its anti-mitotic action by a rapid depletion of the cellular content of guanine nucleotides . Although MPA is a potent inhibitor of GTP synthesis in the HT29 line of human colorectal adenocarcinoma cells in short-term culture, its ability to depress the cloning efficiency of these cells was found to be markedly less than against the mouse mammary carcinoma line, EMT6 . In vivo, MPA is efficiently converted to the biologically inactive O-glucuronide derivative thereby limiting its effectiveness as an anti-tumor agent . Investigation of the fate of MPA incubated with monolayer cultures of HT29 and EMT6 cells revealed that the compound is rapidly converted to the O-glucuronide derivative by HT29 cells, but not by EMT6 cells . Confirmation of the identity of the glucuronide formed by HT29 cells was obtained by its conversion to MPA after incubation with beta-glucuronidase and by comparison of the mass spectrum of its HPLC peak with that of synthetic MPA O-glucuronide . Cultures of two other lines of human colorectal adenocarcinoma cells, Colo-205 and LoVo, also depleted their culture media of MPA although we have not yet established whether these cells also synthesize the glucuronide . The intrinsic partial resistance of HT29 cells to MPA appears to be associated with the ability of these cells to convert MPA to the biologically inactive glucuronide . These results, in conjunction with other reports of the capacity of colorectal cancer cells for Phase I and II metabolism of xenobiotics, may have implications for the design of drugs intended for the treatment of colorectal cancer. Acta Neuropathol (Berl), 1995, 89(5), 431 - 7 Myelin basic protein does not have a mitogenic effect on adult oligodendrocytes; Moore GR et al.; Increased numbers of oligodendrocytes and remyelination are frequently observed in multiple sclerosis plaques . It is presumed the increased numbers of oligodendrocytes are due to cell division, but this has not been proven . The mitogens within the lesion which might be responsible for this are unknown . Since oligodendrocyte proliferation occurs in areas in which there is myelin breakdown, we undertook the present study to determine if myelin basic protein (MBP) or its breakdown products could induce oligodendrocyte proliferation . MBP, or MBP digested by the neutral proteinase plasmin, was added in three concentrations to the media of adult bovine oligodendrocytes in culture . Oligodendrocytes were identified by staining for galactocerebroside . Bromodeoxyuridine incorporation was used as a measure of cell division . Oligodendrocytes were found to divide only rarely in regular culture media, in the presence of MBP, plasmin, or MBP digested by plasmin . The results indicate that MBP is not a significant mitogen for the mature oligodendrocyte. J Immunol, 1995 Jan 1, 154(1), 116 - 27 Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity; Ling P et al.; IL-12, a heterodimeric cytokine, consists of two disulfide-linked subunits, p40 and p35 . We investigated the role of p40 in ligand binding and signal transduction by expressing this subunit alone in COS cells . Culture media of the transfected COS cells exhibited specific dose-dependent binding to KIT225/K6 cells, a human T cell line that expresses IL-12R . Analysis of the culture media by SDS-PAGE and Western blotting demonstrated the presence of 40-kDa monomers and 80-kDa disulfide-linked homodimers . The two p40 species were purified and identified by N-terminal sequencing and proteolytic peptide mapping . Characterization of the p40 proteins for binding and bioactivity showed that both the p40 monomer and dimer inhibited 125I-labeled IL-12 binding to IL-12R, but the 80-kDa species, having a 50% inhibitory concentration (IC50) of 20 to 70 ng/ml, was at least 20-fold more effective than the monomer . Although neither the monomer nor the dimer stimulated human PHA-blast proliferation, the 80-kDa dimer inhibited IL-12-induced proliferation in a dose-dependent manner with an IC50 of 65 ng/ml . The results suggest that the IL-12 p40 subunit contains the essential epitopes for receptor binding . However, a proper conformation required for high affinity binding is achieved only when p40 is associated with a p35 subunit or another p40 subunit . When p40 is associated with a p35 subunit, the heterodimer acts as an agonist mediating biologic activity . However, when p40 associates with another p40, the homodimer behaves as an antagonist in vitro. Microbios, 1995, 83(334), 49 - 57 Isolation, cultivation and partial characterization of a kinetoplastid flagellate from the hindgut of the water bug, Lethocerus indicus; De A et al.; A new species of kinetoplastid flagellates was isolated from the hindgut of the giant water bug, Lethocerus indicus, and successfully cultured in modified glycerol beef extract medium . Light and electron microscopic studies as well as growth in various culture media are described . The flagellates were in three different shapes and sizes in the hindgut of the host as well as in the culture medium . Each form of the parasite contained a kinetoplast which was located near the basal body of the flagellate . The ultrastructural features of this species exhibit great similarity to other species of Bodo . The flagellate ingested bacteria, grew in culture, and was identical with that observed in the hindgut of the bugs . The name Bodo indica has been proposed for this kinetoplastid flagellate. Cancer Res, 1994 Dec 15, 54(24), 6517 - 25 Pericellular pH affects distribution and secretion of cathepsin B in malignant cells; Rozhin J et al.; Redistribution of lysosomes to the cell surface and secretion of lysosomal proteases appear to be general phenomena in cells that participate in local proteolysis . In the present study, we have determined whether malignant progression affects the intracellular distribution and secretion of the lysosomal protease cathepsin B in three model systems, each of which consists of cell pairs that differ in their degree of malignancy . The intracellular distribution of vesicles staining for cathepsin B was evaluated by immunofluorescent microscopy and the secretion of cathepsin B was evaluated by two complementary techniques: stopped assays of activity secreted into culture media; and continuous assays of activity secreted from viable (> or = 95%) cells growing on coverslips . We observed that the intracellular distribution of cathepsin B+ vesicles was more peripheral in the cells of higher malignancy in all three model systems and that active cathepsin B was secreted constitutively from these cells . Because an acidic pericellular pH has been shown to induce translocation of lysosomes in macrophages and fibroblasts, we evaluated the intracellular distribution of cathepsin B+ vesicles and secretion of cathepsin B in cell pairs incubated at slightly acidic pH . Acidic pericellular pH induced a redistribution of cathepsin B+ vesicles toward the cell periphery . In the more malignant cells, this resulted with time in reduced intracellular staining for cathepsin B and enhanced secretion of active cathepsin B . Translocation and secretion of cathepsin B were dependent on a functional microtubular system . Both the redistribution of cathepsin B+ vesicles toward the cell surface induced by acidic pH and the constitutive and acidic pH-induced secretion of active cathepsin B could be inhibited by microtubule poisons and stabilizers . We suggest that the redistribution of active cathepsin B to the surface of malignant cells and its secretion may facilitate invasion of these cells. Biochemistry, 1994 Dec 13, 33(49), 14723 - 32 Characterization of neuronal nitric oxide synthase and a C415H mutant, purified from a baculovirus overexpression system; Richards MK et al.; Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide (.NO) . A baculovirus overexpression system has been developed for a constitutive NOS isoform, cloned originally from rat cerebellum (B-NOS) . Recombinant virus was used at a multiplicity of infection of 5 to infect Spodoptera frugiperda cells in culture, and NOS was expressed to 10% of the total soluble protein at 48 h postinfection . In order to express catalytically active enzyme, it was necessary to supplement the culture media with hemin . This increased the activity of the enzyme 7-fold . A two column affinity purification was developed for the recombinant enzyme, which gave homogeneous protein that migrated at 150 kDa on a denaturing polyacrylamide gel . A Km for L-arginine was determined to be 2.0 +/- 0.4 microM . As isolated, recombinant B-NOS exhibited a Soret maximum at 402 nm, which shifted to 394 nm in the presence of L-arginine . The Soret maximum of the reduced enzyme in the presence of CO was 444 nm . Initial rate steady-state kinetic analysis of the recombinant B-NOS showed evidence of substrate inhibition by L-arginine, which could also be seen in a partially purified preparation of B-NOS from rat cerebella . This substrate inhibition was not observed with the inducible isoform of NOS, purified from immunostimulated murine macrophages . A C415H mutant was overexpressed and purified using the same conditions established for the wild-type recombinant B-NOS . This C415H mutant exhibited no activity and did not bind heme, providing the first experimental evidence to support previously reported primary amino acid comparisons which suggest that C415 provides the coordinating thiolate to the heme moiety in B-NOS. J Mol Biol, 1994 Dec 2, 244(3), 269 - 78 Dependence of lactose metabolism upon mutarotase encoded in the gal operon in Escherichia coli; Bouffard GG et al.; A new gene (galM) has been identified as the fourth cistron of the gal operon, encoding enzymes for the metabolism of galactose and lactose in Escherichia coli . Induction of the gal operon either from the gal promoters or from a neighboring prophage lambda promoter expresses the galM gene as well . The new structure of the gal operon from the promoter end is galE-galT-galK-galM in counter-clockwise orientation on the chromosome . Genetic and biochemical analyses have revealed that the galM gene product has mutarotase activity, which converts alpha-aldose to the beta-anomer . Unlike mutarotase from other bacteria in which the enzyme is primarily processed for export and secretion, the mutarotase from E . coli does not appear to be processed and yet is still found in periplasm (and culture media when overexpressed) in significant amounts . Although the interconversion of the sugar anomers occurs spontaneously in pure water in vitro, the in vivo formation of alpha-D-galactopyranose (the substrate for phosphorylation) from beta-D-galactopyranose (generated by beta-galactosidase hydrolysis of lactose) is largely dependent upon the presence of the mutarotase . This shows that efficient lactose metabolism requires mutarotase . These results give credence to the idea that the activity of intracellular water is not high enough to permit a simple extrapolation of observed in vitro reactions to in vivo situations in every case. J Clin Endocrinol Metab, 1994 Dec, 79(6), 1625 - 31 Production and characterization of endothelin released by human endometrial epithelial cells in culture; Marsh MM et al.; This study identified and characterized endothelin (ET) produced by human endometrial epithelial cells cultured under serum-free conditions, compared the ET released by cells derived from proliferative and secretory phase endometrium, and examined the regulation of ET released by these cells . ET messenger RNA was detected in normal human endometrium with maximal expression in the mid-late secretory phase . Immunoreactive ET released into culture media by separated endometrial epithelial and stromal cells was almost entirely of epithelial cell origin, consistent with the previous immunohistochemical findings . This was identified as ET-1 by reverse phase high-pressure liquid chromatography, and the fractionated conditioned media exhibited bioactivity similar to that of standard ET-1 . Mean ET production was greater from cells derived from proliferative phase endometrium cultured either in serum (P < 0.02) or serum-free conditions (P < 0.02) . Fetal calf serum stimulated ET-1 production from epithelial cells in a dose-responsive manner . ET production was also stimulated by transforming growth factor-beta 1 (2, 5 & 10 ng/mL) and IL-1 alpha (10 & 100 IU/mL) under serum-free conditions but always to a lesser extent than stimulation by serum . The production of ET in human endometrium underlines a potential role for ET in endometrial function. Arch Dermatol, 1994 Dec, 130(12), 1521 - 9 Antiepiligrin cicatricial pemphigoid . A subepithelial bullous disorder; Domloge-Hultsch N et al.; BACKGROUND: Epiligrin is a glycoprotein complex deposited in extracellular matrix by cultured human keratinocytes that serves as the major integrin ligand of these cells . In human skin, epiligrin is found at the interface of the lamina lucida and lamina densa in epidermal basement membrane where it is believed to be associated with anchoring filaments and plays an important role in keratinocyte adhesion . METHODS AND RESULTS: We have identified six patients with a subepithelial bullous disorder of mucous membranes and skin who have IgG anti-basement membrane autoantibodies that immunoprecipitate epiligrin from human keratinocyte extracts and culture media . These patients' IgG autoantibodies also bind epiligrin in human keratinocyte extracellular matrix and epidermal basement membrane as determined by immunofluorescence and immunoelectron microscopy . Studies of 10 patients who are clinically indistinguishable from subjects with anti-epiligrin autoantibodies (ie, cicatricial pemphigoid patients) found that while seven had anti-basement membrane autoantibodies, the latter are directed exclusively against a region of epidermal basement membrane that does not contain epiligrin, are present in low titer (ie, < or = 1:10), do not react with keratinocyte extracellular matrix, and do not bind epiligrin (or any other specific antigen) in immunoprecipitation studies of human keratinocyte extracts or media . Antiepiligrin autoantibodies were also not detected in studies of 36 additional patients with bullous diseases or six normal volunteers . CONCLUSIONS: Cicatricial pemphigoid is a disease phenotype in which patients' autoantibodies may target different constituents of epidermal basement membrane . Antiepiligrin autoantibodies are a specific immunologic marker for a group of patients with a disease entity that we propose to designate antiepiligrin cicatricial pemphigoid. Exp Cell Res, 1994 Dec, 215(2), 338 - 46 Regulation of fibronectin by insulin-like growth factor-I in cultured rat thoracic aortic smooth muscle cells and glomerular mesangial cells; Tamaroglio TA et al.; We examined the regulation of fibronectin (FN) levels by insulin-like growth factor-I (IGF-I) in rat thoracic aortic smooth muscle cell (SMC) and glomerular mesangial cell cultures . IGF-I enhances FN levels in the culture media of the SMC and in the cell samples of the mesangial cells . It shows no effect on FN levels in the cell samples of the SMC and in the culture media of the mesangial cells . Modulation of IGF-I-induced FN levels by insulin was also examined . Insulin enhances FN levels in the culture media of the SMC but not in the cell samples . Insulin did not induce an increase in FN levels in either the mesangial cell samples or the culture media . No additive effect of FN levels was observed when the SMC were treated with insulin and IGF-I together . The effect of IGF-I on FN mRNA levels was assessed . IGF-I enhances SMC and FN mRNA levels, implying that acquisition of additional FN mRNA units accounts for the increase in FN levels . The effects of actinomycin D and cycloheximide on IGF-I-induced FN mRNA levels were also examined . Inhibition of the IGF-I-induced FN mRNA levels by actinomycin D and an increase of FN mRNA levels by cycloheximide suggest that IGF-I regulates FN mRNA synthesis at the transcriptional level. Mol Cell Endocrinol, 1994 Dec, 106(1-2), 121 - 30 Expression and characterization of recombinant rat placental prolactin-like protein C; Conliffe PR et al.; Prolactin-like protein C (PLP-C) is a member of the rat placental family of proteins which are structurally related to pituitary prolactin (PRL) . In an effort to characterize the receptor specificity and biological activity of PLP-C, we used a PLP cDNA to express the recombinant protein in a bacterial system . The PLP-C cDNA was modified by oligonucleotide mutagenesis and ligated into a human carbonic anhydrase II (hCAII) expression vector . Following a single step affinity purification, the hCAII-PLP-C fusion protein was digested with enterokinase to release a 25 kDa protein . N-Terminal sequence analysis of the 25 kDa band demonstrated identity with PLP-C . A polyclonal antiserum to the fusion protein cross reacted with seven major proteins in rat placental culture media of which two were the native forms of PLP-C . Recombinant PLP-C was not mitogenic in the Nb2 lymphoma bioassay and did not exhibit high affinity binding to rat PRL receptor . The choice of hCA-II fusion allows for rapid purification of rPLP-C which will aid in further investigation of the biological role of PLP-C. Hum Mol Genet, 1994 Dec, 3(12), 2223 - 9 Toward understanding the pathogenic mechanisms in gelsolin-related amyloidosis: in vitro expression reveals an abnormal gelsolin fragment; Paunio T et al.; Gelsolin-related amyloidosis, also called familial amyloidosis, Finnish type (FAF) is an autosomal dominantly inherited disorder characterized by progressive polyneuropathy and corneal lattice dystrophy . All the analyzed patients are found to carry a nucleotide substitution of A or T for G654 in their gelsolin gene, which at the protein level results in the conversion of the 187 amino acid residue, aspartic acid, to asparagine or tyrosine, respectively . In this study, we transfected mammalian mesenchymal COS-1 cells with a derivative of the expression vector pCD-X containing cDNA coding for the wild-type (D187) and mutant forms (N187 and Y187) of plasma gelsolin . Both disease-associated mutant forms of gelsolin were found to be abnormally processed, which led to the secretion of an aberrant 68 kDa gelsolin fragment into the culture media . This fragment most probably represents a carboxy-terminal part of the protein and contains the suggested amyloid-forming sequence . Initial data were also obtained for involvement of a metalloendoprotease in the pathologic processing . This aberrant proteolysis is likely to represent a crucial initiator step in the cascade resulting in amyloid accumulation in patients' tissues. Plant Mol Biol, 1994 Dec, 26(5), 1521 - 8 Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development; Walden R et al.; Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis . The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype . While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines . The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue . Confirmation that the genomic sequence responsible for the selected phenotype has indeed been isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions . Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis . This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated. FEMS Microbiol Rev, 1994 Dec, 15(4), 321 - 53 Notes on protozoa in agricultural soil with emphasis on heterotrophic flagellates and naked amoebae and their ecology; Ekelund F et al.; Heterotrophic flagellates and naked amoebae are usually very numerous in agricultural soils; with numbers in the magnitude of 10,000 to 100,000 (active+encysted) cells per gram of soil . In 'hotspots' influenced by living roots or by dead organic material, the number may occasionally be as high as several millions per gram of soil . An exact enumeration of these organisms is virtually impossible . As they most often adhere closely to the soil particles, direct counting will underestimate numbers since the organisms will be masked . The method usually applied for enumeration of these organisms, the 'most probable number (MPN) method', is based on the ability of the organisms to grow on particular culture media . This method will in many cases underestimate the total protozoan number (active+encysted) . It is uncertain how many of the heterotrophic flagellates and naked amoebae are actively moving and how many are encysted at a particular time; the 'HCl-method' which has usually been used to discriminate between active and encysted has proven to be highly unreliable . Despite the methodological difficulties many investigations of these organisms indicate that they play an important role in agricultural soils as bacterial consumers, and to a minor extent as consumers of fungi . Because of their small size and their flexible body they are able to graze bacteria in small pores in the soil in which larger organisms are precluded from coming . Key factors restricting the number and activity of heterotrophic flagellates and naked amoebae in soils seem to be water potential and soil structure and texture . In micro-cosm experiments, small heterotrophic flagellates and naked amoebae regulate the size and composition of the bacterial community . Bacterial activity seems to be stimulated by these organisms in most cases as well as the mineralization of carbon and nitrogen and possibly other mineral nutrients . In the rhizosphere of living plants the activity of protozoa has proven to stimulate uptake of nitrogen in pot experiments, and it has been hypothesized that organic matter liberated by plants in the root zone will stimulate bacterial and protozoan activity, leading to mineralization of organic soil nitrogen which is subsequently taken up by the plants. J Egypt Soc Parasitol, 1994 Dec, 24(3), 611 - 9 Evaluation of two cultural media (CPLM & TYM) for isolation and maintenance of Trichomonas vaginalis stocks in the laboratory; Abd el Ghaffar FM et al.; Five hundreds vaginal discharge specimens were inoculated simultaneously in 2 axenic culture media (CPLM & TYM), in order to compare their ability to isolate and to maintain the growth of T . vaginalis in the laboratory . While both media were found to be equally good in detecting the organisms in vaginal discharges, yet, T . vaginalis stocks were maintained for a longer time in TYM medium (one year), than in the CPLM medium (2 weeks) . The yields of the parasites with different inocula subcultured and after different incubation periods were counted in the TYM medium. Naturwissenschaften, 1994 Dec, 81(12), 528 - 35 Radio-frequency microtools for particle and liver cell manipulation; Fuhr G et al.; Single particles can be manipulated by applying high frequencies to ultramicro electrode arrays fabricated on planar structures . Heat production can be reduced to the extent that intense electric fields can be applied even to unmodified cell culture media . Animal cells grow normally in the high field (up to 100 kV/m) between such continuously energized multielectrodes . As with laser tweezers {1-3}, this technique can capture particles and cells in field traps, generate linear movement, and permit cell cultivation . It can also produce micropatterns of pH gradients, field-cast objects, and control cell adhesion . These microtools may be combined to develop cell separators, microsensors, and controlled-biocompatibility surfaces. Bioseparation, 1994 Dec, 4(6), 369 - 81 Comparison of membrane adsorber (MA) based purification schemes for the down-stream processing of recombinant h-AT III; Reif OW et al.; Several multistage chromatographic separation schemes based on Membrane Adsorbers as stationary phases were designed for the isolation of recombinant human Antithrombin III from supernatants of baby hamster kidney cell cultures . The Antithrombin III concentration of the culture supernatants varied between 6 micrograms/ml and 14 micrograms/ml . The culture media contained 10% foetal calf serum . The concomitant overall protein concentration ranged from 5.4 mg/ml to 6.2 mg/ml, with bovine serum albumin constituting approximately 60% . Strong cation and anion exchanger, Heparin-, and Cibacron Blue Membrane Adsorber were used . While Heparin-Membrane Adsorber were found to isolate and concentrate the Antithrombin III efficiently, the removal of the major bovine serum proteins (albumin, transferrin, immunoglobulins) required a multistage process . By using a sequence of ultrafiltration, diafiltration, Cibacron Blue, anion exchanger, and Heparin-Membrane Adsorber, an electrophoretically pure Antithrombin III could be obtained . Subsequent high sensitivity gel immunoelectrophoresis proved the isolated protein free of bovine IgG and bovine transferrin, while approximately 2% serum albumin could still be discerned . A reduction of the serum content (3%) allowed the isolation of high purity Antithrombin III (> 99.9%), however, the product's specific activity was halved . The Down-Stream-process was designed and optimised for a mobile phase flow rate of 2 ml/min (0.12 l/h) . With the exception of the final Heparin affinity step, however, flow rates of up to 4.8 l/h could be used without adverse effect on the final purity, with a concomitant increase of the throughput . Batches of up to 10 1 cell culture supernatant were processed in an automated procedure. Pigment Cell Res, 1994 Dec, 7(6), 419 - 27 Melanins in IGR 1 melanoma cells; Odh G et al.; Information on the composition of melanins is obtained by analysis both of 4-amino-3-hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole-2,3,5-tricarboxylic acid (PTCA) after potassium permanganate oxidation . Analysis of thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3-dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups . Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin . The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups . We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line . Cell cultures were harvested after 2, 4, and 7 days . Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days . Cells without medium change had seven times the amount of melanin found in cultures with medium change . The PDCA/PTCA ratio decreased with increasing amounts of melanin . With increased melanization, eumelanin is increased relatively more than pheomelanin . The cell content of 5-S-cysteinyldopa (5-S-CD) was similar in all cultures, while 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures. Cell Biol Int, 1994 Dec, 18(12), 1105 - 14 Co-culture, oviduct secretion and the function of oviduct-specific glycoproteins; Nancarrow CD et al.; Co-cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts . Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development . Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media . Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media . These glycoproteins are induced by oestrogen in vivo but not in vitro . It is our contention that co-cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells. Cell Biol Toxicol, 1994 Dec, 10(5-6), 361 - 5 Influence of fibroblasts on epidermization by keratinocytes cultured on synthetic porous membrane (insert) at the air-liquid interface; Robert M et al.; Culture of keratinocytes on a noncoated porous synthetic membrane maintained at the air-liquid interface allows the establishment of a fibroblast/keratinocyte co-culture, without direct cell-cell contact between the two cellular layers . The influence of fibroblasts (proliferating, confluent or blocked by mitomycin C) on epidermization (i.e., expression of integrins and markers of epidermal differentiation) was studied by immunohistochemistry in two culture media . In the medium supplemented with FCS or Ultroser G and in the absence of fibroblasts, alpha 2, alpha 3, alpha 5 and alpha 6 subunits of integrins are expressed by the basal keratinocytes, except alpha 5 which does not appear with the medium supplemented with Ultroser G . During stratification, the alpha 3 subunit is the only one to persist on suprabasal cells and all the markers of epidermal differentiation studied (filaggrin, involucrin, transglutaminase, keratins K1/K10) are expressed at the 14th day of emerged culture . The presence of fibroblasts modifies the expression profile of integrins: when they are proliferative, the expression of alpha 2 and alpha 6 chains is delayed in the medium supplemented with FCS, and the alpha 6 chain is absent in the medium supplemented with Ultroser G; when they are confluent or blocked by mitomycin C, greater changes are observed only in the medium supplemented with Ultroser G and lead to inhibition or delay of the expression of alpha 2 and alpha 6.(ABSTRACT TRUNCATED AT 250 WORDS) Biol Reprod, 1994 Dec, 51(6), 1145 - 53 A novel glycoprotein of the aspartic proteinase gene family expressed in bovine placental trophectoderm; Xie S et al.; The pregnancy associated glycoproteins (PAG 1) that appear in the maternal serum of cattle and sheep soon after implantation are apparently inactive members of the aspartic proteinase family . Here we describe the isolation of a highly abundant cDNA (PAG 2 cDNA) that represents a second member of this gene family which is structurally related to bovine PAG 1, ovine PAG 1, and pepsin (58%, 58%, and 51% amino acid sequence identity, respectively) . The bovine PAG 2 cDNA was identified in two ways . First, the bovine placental library was screened under relatively nonstringent conditions with an ovine PAG 1 cDNA . The second fortuitous approach employed immunoscreening with an antiserum raised against a partially purified factor that competed with bovine LH for binding to the LH receptor on the CL of the ovary . The full-length cDNA (1258 bp) codes for a polypeptide of 376 amino acids . Bovine PAG 2, unlike bovine PAG 1, has a catalytic center with a consensus sequence of amino acids . Its mRNA is expressed in fetal placenta but not in other fetal organs, and is localized to both the mononucleate and binucleate cells of the trophectoderm, whereas PAG 1 is expressed only in binucleate cells . PAG 2 is synthesized by placental explants as a 70-kDa glycoprotein that is processed to several smaller molecules . Western blot analysis of culture media developed with epitope-selected antibodies to PAG 2 reveals several bands ranging in apparent M(r) from 31,000-70,000, which correspond in size to the polypeptides present in the preparation used for immunization.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1994 Nov 30, 205(1), 221 - 9 Processing of human prosomatostatin in AtT-20 cells: S-28 and S-14 are generated in different secretory pathways; Brakch N et al.; Somatostatin-14 (S-14) and somatostatin-28 (S-28) are generated by differential processing of a single precursor at a dibasic (R-K) or monobasic (R) proteolytic cleavage site, respectively . To study the pathways of processing of prosomatostatin, we have expressed in AtT20 cells cDNA encoding human prosomatostatin and prosomatostatin mutated in one or the other processing site . Analysis of the peptides present in cell extracts or culture media before and after stimulation of the cells with 8-BrcAMP indicated that prosomatostatin can enter three distinct secretory pathways where it is differently processed: 1) prosomatostatin was secreted through the constitutive pathway; 2) the regulated secretory pathway generated S-14 which was released upon stimulation of the cells with 8-BrcAMP; 3) an alternative pathway, insensitive to 8-BrcAMP produced S-28 and S-14 . Moreover, our results suggest that the R-K processing site used to produce S-14 is an important structural feature for targeting the precursor to the regulated secretory pathway. Biochem Biophys Res Commun, 1994 Nov 15, 204(3), 1305 - 11 Nitric oxide production by cells isolated from regenerating rat liver; Obolenskaya M et al.; Nitric oxide (NO) production by cells of the regenerating liver was estimated from the amount of nitrite accumulated during 24 h in the culture media of hepatocytes, Kupffer cells and sinusoidal endothelial cells isolated at different times after partial hepatectomy (PHE) . The time course of NO production was compared with the course of the proliferating activity of the same cells . During the time when liver cells pass through their first cell cycles, hepatocytes were the main producers of NO in the liver . The time-dependent changes of their NO production corresponded to those obtained with the whole liver and were inversely correlated with the DNA-synthesizing activity . The NO production by Kupffer and endothelial cells followed that by hepatocytes in this order; the time displacement between them corresponded to the schedule of their proliferating activity . The NO synthesis in non-parenchymal cells fluctuated in a similar way as in parenchymal cells and was minimal when DNA synthesis was manifest. FEBS Lett, 1994 Nov 7, 354(2), 232 - 6 Regulatory effects of galactose on galactose-1-phosphate uridyltransferase activity on human hepatoblastoma HepG2 cells; Davit-Spraul A et al.; Galactose-1-phosphate uridyltransferase (GALT) deficiency results in galactosemia in man . We have studied the regulation of the GALT gene expression on the HepG2 cell line by growing the cells in glucose or galactose medium . No difference of Km values was observed in glucose or galactose media but the Vmax value with galactose was 50% higher than that with glucose . Also in galactose medium, an increased GALT specific activity was detected suggesting the production of more enzyme proteins . Yet, slot dot quantification of GALT mRNA revealed a decreased amount of these transcripts in cells cultured with galactose or inosine while Northern blot analysis revealed the normal 1.4 kb transcript in all culture media used . Finally, IEF gel analysis displayed different isozymic patterns for the GALT enzyme in cells grown in glucose, galactose or inosine media . With glucose-free media, the major band of GALT corresponds to that found in human liver . Altogether, these results suggest that the control of GALT gene expression in HepG2 cells is located at the post-transcriptional level and correlated to the growth rate of the cell. Toxicol Appl Pharmacol, 1994 Nov, 129(1), 12 - 5 Influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on TNF-alpha levels in the skin of congenic haired and hairless mice; Connor MJ et al.; It has been proposed that TNF-alpha mediates TCDD-induced toxicity . TCDD induces a chloracne-like response in the skin of hairless HRS/J mice but not in congenic haired animals . Using an ELISA, we measured TNF-alpha levels in the skin of TCDD-treated haired and hairless HRS/J mice to test the hypothesis that TNF-alpha mediates the cutaneous toxicity of TCDD . TNF-alpha levels in the skin of haired mice were at or below minimal detectable levels and were unchanged by TCDD exposure . In contrast, TNF-alpha levels were significantly higher in the skin of hairless mice after TCDD exposure . The bulk of the induced TNF-alpha was present in the dermis, although detectable amounts were present in the epidermis . To determine if murine skin cells were producing TNF-alpha in direct response to TCDD, cultures of neonatal epidermal keratinocytes and dermal fibroblasts were treated with varying biologically active doses of TCDD or vehicle (DMSO) or with lipopolysaccharide (LPS) as a positive control . Within 24 hr of exposure to LPS, TNF-alpha levels were increased in the culture media of all cells tested . In contrast, TCDD treatment (10(-11) M to 10(-7) M) failed to induce detectable TNF-alpha release from either fibroblasts or keratinocytes over a comparable time frame or when measured for up to 6 days following exposure . The failure of TCDD to stimulate TNF-alpha production by keratinocytes or fibroblasts suggests that the rise in dermal TNF-alpha levels seen in vivo is unlikely to be a primary component of the mechanism of toxicity . We suggest that the source of the dermal TNF-alpha in TCDD-treated hairless mouse skin is probably component cells of the inflammatory response. Exp Cell Res, 1994 Nov, 215(1), 237 - 9 Derivation of germline competent embryonic stem cells with a combination of interleukin-6 and soluble interleukin-6 receptor; Nichols J et al.; In this report we document the derivation of pluripotential embryonic stem (ES) cells in the absence of a feeder layer by supplementation of culture media with either ciliary neurotrophic factor or oncostatin M, or with a combination of interleukin-6 (IL-6) plus soluble interleukin-6 receptor (sIL-6R) . These factors all activate gp130-associated signaling processes, as does the previously characterized ES cell maintenance factor Differentiation Inhibiting Activity (Leukemia Inhibitory Factor) . In particular, the IL-6/sIL-6R complex is thought to act exclusively through gp130 . All ES cell lines derived using IL-6/sIL-6R contributed extensively to chimeras and were transmitted through the germline at high frequency . These findings point to a pivotal role for gp130 in ES cell propagation and may be relevant to attempts to derive ES cells from species other than mouse. J Immunol, 1994 Nov 1, 153(9), 4048 - 58 Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase; Wang J et al.; This report demonstrates that incubation of cytotoxic T cells with NAD causes suppression of their ability to proliferate in response to stimulator cells or to lyse targets . Effects are evident after incubation for 3 h with concentrations of NAD as low as 1 microM and are sustained for many hours after removal of NAD from culture media . Suppression is a result of the failure of CTL to form specific conjugates with targets as well as a lower level of activation in response to TCR-mediated stimulation, although TCR-mediated transmembrane signaling is demonstrable . Metabolites of NAD such as nicotinamide, ADP-ribose, and cyclic-ADP-ribose have no detectable effect, indicating that NAD-glycohydrolase or ADP-ribose cyclase do not mediate suppression . Incubation of intact CTL with {32P}NAD leads to incorporation of 32P into a particulate, subcellular fraction, a reaction that is not inhibitable by ADP-ribose . Hydroxylamine, but not mercuric ion releases {32P}ADP-ribose, whereas phosphodiesterase releases {32P}AMP from the particulate subcellular fraction, suggesting that labeling is a result of enzymatic mono-ADP-ribosylation of arginines . In support of this, treatment of intact CTL with phosphatidylinositol-specific phospholipase C releases an arginine-specific ADP-ribosyltransferase and causes insensitivity to ecto-NAD suppression . These results suggest that a GPI-anchored ADP-ribosyltransferase uses ecto-NAD to ADP-ribosylate proteins that regulate CTL function. Invest Radiol, 1994 Nov, 29(11), 955 - 62 Effects of iodinated x-ray contrast media on renal epithelial cells in culture; Andersen KJ et al.; RATIONALE AND OBJECTIVES . To study cellular mechanisms that cause contrast media nephropathy, an in vitro system for proximal and distal tubular cells has been established to evaluate the influence of x-ray contrast media on tubular function . METHODS . Confluent cell cultures of the two renal cell lines, proximal tubule (LLC-PK1) and distal tubule (MDCK), were exposed for 20 hours to 0 to 100 mg iodine/mL of the ionic monomer metrizoate, the ionic dimer ioxaglate, and the non-ionic monomer iohexol . Toxicity was assessed by electron microscopy, cell viability, and biochemical assays of brush-border and lysosomal marker enzymes . RESULTS . The results demonstrated a concentration-dependent toxic effect from the contrast media on cellular appearance consisting of an increased vacuolization and on the activity of brush-border and lysosomal marker enzymes in cells and in culture media . CONCLUSION . The results, in which the nonionic x-ray contrast media iohexol appeared to be less toxic than the ionic x-ray contrast media investigated, demonstrated that defined renal cells in culture are valuable tools in studies regarding renal toxicity of x-ray contrast media. Pharmacol Toxicol, 1994 Nov, 75(5), 310 - 4 Derivatives of 2-nitrofluorene cause changes of human sperm motility; Leijonhufvud PK et al.; The effects on human sperm motility characteristics of 2-nitrofluorene and selected derivatives were studied in vitro, using computer aided sperm analysis (Cellsoft) . Substances to be tested were dissolved in acetone and added to separated spermatozoa in culture media to final concentrations of 100 and 1000 microM . Aliquots were removed immediately (< 5 min.) and 24 hr after the addition and tested for sperm motility characteristics . Four of the substances tested; 2,4,7-trinitrofluoren-9-one (2,4,7-tNFO), 2,5-diaminofluorene (2,5-dAF), 7-hydroxy-2-nitrofluorene (7-OH-NF) and 2,7-diaminofluorene (2,7-dAF) showed strong detrimental effects on the sperm motility . Slight detrimental effects were also recorded using 2-nitrofluorene and 2,5-dinitrofluorene (2,5-dNF) . Weak stimulatory effects were obtained using 2-acetoamidofluorene (AAF) and 2,7-dinitrofluorene (2,7-dNF) . No significant effects were seen with 5-hydroxy-2-nitrofluorene (5-OH-NF), 2-aminofluorene (AF), 2-aminofluoren-9-one (AFO), 2-amino-9-hydroxyfluorene (9-OH-AF) or 9-hydroxy-2-nitrofluorene (9-OH-NF) . The mechanism behind this effect is not known but it could be speculated that these lipophilic substances interact with the membranes or the cellular respiration. J Dermatol, 1994 Nov, 21(11), 838 - 46 Antibody-binding to the 180-kD bullous pemphigoid antigens at the lateral cell surface causes their internalization and inhibits their assembly at the basal cell surface in cultured keratinocytes; Kitajima Y et al.; We demonstrated the effects of monoclonal antibodies to the 180-kD and 230-kD BP antigens (BPA) and of BP sera on Ca(++)-induced formation of hemidesmosomes in cultured human keratinocytes (a cell line, DJM-1) by immunofluorescence microscopy . Under low Ca++ (0.07 mM) conditions, the 180-kD and 230-kD BPAs were distributed homogeneously on the basal plasma membrane, while they formed a peculiar concentric ring or arch (ring/arch) arrangement in high-Ca++ (1.87 mM) medium . On the other hand, the apical-lateral cell membrane was stained homogeneously with antibodies to the 180-kD BPA, but not to the 230-kD BPA, both in low and high Ca++ media . The low-high Ca++ switch at first caused disappearance of the antigen from the basal plasma membrane and then formed the high-Ca++ ring/arch pattern within 3 hrs . In this system, monoclonal antibodies to the 180-kD and 230-kD BPAs and the sera from 5 BP patients, 2 pemphigus vulgaris (PV) patients, and 4 normal volunteers were added into the culture media . The addition of anti-180-kD BPA antibodies or any BP serum caused the internalization of the 180-kD BPA from the apical-lateral cell membrane and inhibited the Ca(++)-induced formation of the ring/arch pattern on the basal membrane, possibly by inhibiting the movement of the antigen from the lateral to the basal membrane to form hemidesmosomes.(ABSTRACT TRUNCATED AT 250 WORDS) Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1994 Nov-Dec, 35(6), 508 - 13 Diagnosis of I-cell disease; Hwu WL et al.; I-cell disease (mucolipidosis II) is a rare lysosomal storage disease, with its primary defect the deficiency of an enzyme responsible for lysosomal enzyme processing, resulting in multiple lysosomal enzyme insufficiency . Diagnosis of I-cell disease usually can be made by the specific patterns of enzyme distribution: deficient intracellular, but excessive extracellular, enzymes . A six month old female infant was found to have bilateral congenital dislocation of hips, developmental delay, coarsening of facial appearance and dysostosis multiplex . In view of the very early onset of disease, I-cell disease was suspected . Lysosomal enzyme tests (including alpha-mannosidase, alpha-fucosidase, beta-glucuronidase and beta-galactosidase) were performed on the leukocytes, skin fibroblasts, plasma and media from fibroblast cultures . All activities of the four enzymes were low in both leukocytes and fibroblasts, but were 10- to 70-fold higher than normal in plasma, and high in culture media . Both the clinical and laboratory findings here were consistent with a diagnosis of I-cell disease. Prep Biochem, 1994 Nov, 24(3-4), 251 - 61 Purification and characterization of lysine- and arginine-specific gingivain proteases from Porphyromonas gingivalis; Bedi GS; Four gingivain proteases, active in presence of L-cysteine, were purified from spent culture media of oral pathogen Porphyromonas gingivalis by ion-exchange chromatography on MonoQ and chromatofocusing on MonoP columns . Three of the purified proteases, with molecular masses of 75 kDa, 70 kDa and 55 kDa, respectively, hydrolyzed synthetic chromogenic substrates with arginine in the P1 position . One protease, with a molecular mass of 80 kDa, hydrolyzed substrates with lysine in the P1 position . It is proposed these enzymes be named: arg-gingivain-75, arg-gingivain-70, arg-gingivain-55, and lys-gingivain-80, respectively, based on their molecular mass and specificity for either arginine or lysine in the P1 position. Ginekol Pol, 1994 Nov, 65(11), 638 - 41 {Synthesis of estradiol by endometrial stroma cells under the influence of epidermal growth factor (EGF) in vitro}; Tomaszewski J et al.; The endometrial samples were obtained from 13 operated women in the proliferative phase of menstrual cycle . The tissue culture of endometrial stromal cells was established . After the cells were growth to the confluency, EGF was added to dishes of study group to the final concentrations of 5 ng/ml . Culture media of study and control groups were collected every 48, 96, 144, 192 hours and lyophylized . The estradiol level was measured in culture media by RIA . The mean estradiol concentrations in culture media of endometrial stromal cells decreased during the time of experiment in the control: 318 pg/ml, 276 pg/ml, 186 pg/ml and 110 pg/ml and study group respectively: 339 pg/ml, 279 pg/ml, 169 pg/ml and 129 pg/ml . There was no significant influence of EGF (5 ng/ml) on estradiol level in culture media in each time intervals. Nippon Rai Gakkai Zasshi, 1994 Nov, 63(3), 75 - 85 Down regulation of Ia expression in macrophages following incubation with mycobacteria; Fukutomi Y et al.; Macrophages are known to release cytokines in response to various kinds of stimulators . In the present study, peritoneal macrophages from C3H/He or C3H/HeJ mice were incubated in vitro with heat-killed M . lepraemurium, M . intracellulare or M . gordonare for 3 days followed by harvest culture supernatant to analyze cytokine activities . It, therefore, seems that macrophages phagocytizing these mycobacteria, released interleukin-1 (IL-1) and tumor necrosis factor (TNF) in culture media . The amount of release was dose dependent on mycobacteria employed . In addition, macrophages, as already have reported elsewhere, treated with IFN for 2 to 3 days showed enhanced expression of surface Ia; although the expression was inhibited if the cells phagocytized mycobacteria . Similarly, the reduced expression of Ia was observed in peritoneal macrophages from MRL/lpr mice after 3 day-culture with mycobacteria in vitro . More importantly, in the presence of the supernatant obtained from macrophages incubated with mycobacteria, IFN gamma-treated normal macrophages exhibited suppressed expression of Ia . These results demonstrate that cytokine release and reduced expression of surface Ia in macrophages are simultaneous phenomena after phagocytosis of mycobacteria . Suppression of Ia may be in part induced by Ia suppressive factor(s) released from mycobacterium-phagocytized macrophages. Presse Med, 1994 Oct 22, 23(32), 1483 - 8 {Atypical mycobacterial infections}; Dautzenberg B et al.; Infrequent and forgotten before the advent of the acquired immune deficiency syndrome (AIDS), non-tuberculous mycobacterial infections are now often encountered, predominately in patients positive for the human immune deficiency virus (HIV) . In non-AIDS patients, Mycobacterium kansasii, M . avium and M . xenopi are the most common causal agents of pulmonary mycobacterial infections . Nodes and skin diseases are less frequent . M . kansasii infections are treated for 12 months with a standard combination of rifampin, isoniazid and ethambutol . The treatment for M . xenopi and M . avium infections have not yet been standardized . The AIDS epidemia has modified the epidemiology of these disease and there has been a 10-fold increase in incidence . Disseminated M . avium infections occur in 15% of patients at end-stage AIDS . This new epidemia has triggered research leading to the discovery of new diagnostic procedure including blood culture media for mycobacteria, polymerase chain reaction (PCR) and new active drugs . New active macrolides such as clarithromycine and azithromycine are active against M . avium and new rifampicin-related drugs such as rifabutine and new quinolones are under investigation. J Biol Chem, 1994 Oct 21, 269(42), 26280 - 5 Type I procollagen COOH-terminal proteinase enhancer protein: identification, primary structure, and chromosomal localization of the cognate human gene (PCOLCE); Takahara K et al.; Type I procollagen COOH-terminal proteinase (C-proteinase) enhancer, a glycoprotein that binds to the COOH-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity, was purified from mouse fibroblast culture media . Partial amino acid sequences obtained from proteolytic fragments were found to have identity with the deduced amino acid sequence of a cDNA clone of unknown function, previously isolated from a mouse astrocyte library . Sequences of mouse enhancer cDNA, obtained in the present study, predict a approximately 50-kDa, 468-amino acid protein that differs from the 43-kDa, 402-amino acid protein predicted by the previously reported astrocyte-derived clone . Human cDNAs encode an enhancer of 449 amino acids . Previous biochemical studies have found the mouse enhancer as a 55-kDa form, which is readily processed to 36- and 34-kDa forms, retaining full C-proteinase enhancing activity and the ability to bind the COOH-terminal propeptide . Data presented here show the 36-kDa form to correspond to the amino-terminal portion of the 55-kDa protein . This is the most conserved region between mouse and human enhancers, comprising two domains with homology to domains found in a number of proteases and proteins with developmental functions . Such domains are thought to mediate interactions between proteins . Mouse enhancer RNA is shown to be at highest levels in collagen-rich tissues, especially tendon . The human enhancer gene, PCOLCE, is localized to 7q21.3-->q22, the same chromosomal region containing the type I collagen alpha 2 chain gene, COL1A2. Biol Trace Elem Res, 1994 Oct-Nov, 46(1-2), 113 - 23 Selenium enhances glutathione peroxidase activity and prostacyclin release in cultured human endothelial cells . Concurrent effects on mRNA levels; Ricetti MM et al.; Selenium (Se) is an essential component of glutathione peroxidase (GSH-Px), an enzyme that protects cells by reducing intracellular peroxides . Impaired Se status and GSH-Px activity seem associated with increased risk of atherosclerotic vascular diseases . This study reports the effects of Se supplementation on GSH-Px activity, on prostacyclin (PGI2) production, on 12-hydroxy-eicosatetraenoic acid (12-HETE) levels, and on GSH-Px mRNA expression in cultured human umbilical vein endothelial cells (HUVEC) . Se-enriched HUVEC showed significant increase of both GSH-Px activity and thrombin-stimulated production of PGI2 in the presence of stable concentrations of 12-HETE . On the other hand, an inverse correlation between Se concentrations in culture media and GSH-Px mRNA levels in Northern blot analysis was shown; this suggests that a major degree of regulation for GSH-Px expression by Se is most likely exerted at the posttranscriptional level . These observations may help to explain the increased incidence of atherosclerosis described in Se-deficient individuals. Environ Health Perspect, 1994 Oct, 102 Suppl 5, 103 - 7 Dissolution of man-made vitreous fibers in rat alveolar macrophage culture and Gamble's saline solution: influence of different media and chemical composition of the fibers; Luoto K et al.; The effect of different chemical compositions of man-made vitreous fibers (MMVF) on their dissolution by alveolar macrophages (AM) in culture and in Gamble's solution was studied . The fibers were exposed to cultured rat AMs, culture medium alone; or Gamble's saline solution for 2, 4, or 8 days . The dissolution of the fibers was studied by measuring the amount of silicon (Si), iron (Fe), and aluminum (Al) in each medium . The AMs in culture dissolved Fe and Al from the fibers but the dissolution of Si was more marked in the cell culture medium without cells and in the Gamble's solution . The dissolution of Si, Fe, and Al was different for different fibers, and increased as a function of time . The Fe and Al content of the fibers correlated negatively with the dissolution of Si by AMs from the MMVF, i.e., when the content of Fe and Al of the fibers increased the dissolution of Si decreased . These results suggest that the chemical composition of MMVFs has a marked effect on their dissolution . AMs seem to affect the dissolution of Fe and Al from the fibers . This suggests that in vitro models with cells in the media rather than only culture media or saline solutions would be preferable in dissolution studies of MMVFs. Am J Reprod Immunol, 1994 Oct, 32(3), 180 - 3 Interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) in peritoneal fluid and macrophage-conditioned media of women with endometriosis; Keenan JA et al.; PROBLEM: The presence of the various cytokines in human peritoneal fluid has been incompletely evaluated . Changes in cytokine levels may be related to activation of peritoneal macrophages, development of endometriosis, and infertility . This study assesses peritoneal fluid levels of interferon gamma (IFN-gamma) and interleukin-6 (IL-6), and peritoneal macrophage production of IL-6, in women with and without endometriosis . METHOD: Peritoneal fluid was obtained from 62 women at the time of diagnostic or operative laparoscopic surgery for benign gynecologic disease . Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected . IFN-gamma and IL-6 levels in peritoneal fluid samples and macrophage conditioned media were determined by commercial ELISA . RESULTS: IL-6 was significantly higher in the macrophage conditioned media of women with endometriosis as compared with controls . IL-6 levels were fourfold higher in early stage endometriosis (P < 0.05) and eightfold higher in advanced endometriosis . There were no significant differences between groups in the peritoneal fluid levels of IL-6 or IFN-gamma . CONCLUSIONS: Peritoneal macrophage IL-6 secretion is increased in women with endometriosis, and appears to correlate with disease stage . IFN-gamma does not appear to be responsible for the activation of macrophages in women with endometriosis. Melanoma Res, 1994 Oct, 4(5), 287 - 91 Levels of dopachrome tautomerase in human melanocytes cultured in vitro; Bernd A et al.; Several reports have been published about the level of activity and possible functions of dopachrome tautomerase (DCT) in mouse melanoma cells . Data about the levels of this activity in human melanocytes in culture are still scarce, and, as far as we know, a comparison between mouse and human melanocytes, or between normal and malignant melanocytes, has never been published . We have measured the tyrosinase and DCT activities, as well as the melanin content, in mouse Cloudman melanoma cells, two lines of human melanoma, and three lines of normal human melanocytes obtained from fetal skin . Although more cell lines should be tested to draw a general conclusion, our results suggest that normal melanocytes contained much higher tyrosinase activity and melanin content but lower DCT activity than malignant melanocytes . The two lines of human melanoma cells tested had lower levels of DCT activity than Cloudman melanoma cells . Finally, the low level of DCT activity found in normal human melanocytes cultured in vitro cannot be explained by any of the necessary stimulatory factors added to the cell culture media. Anal Biochem, 1994 Oct, 222(1), 29 - 33 Purification of humanized murine and murine monoclonal antibodies using immobilized metal-affinity chromatography; Hale JE et al.; An affinity purification technique has been developed using mild elution conditions for the isolation of humanized murine and murine IgG1 . This technique is based on the innate affinity of IgG1 for metal and utilizes immobilized metal-affinity chromatography . Antibody bound to the metal-affinity column is eluted with a descending pH gradient and IgG1 elutes around pH 6.0 . Humanized murine IgG1 isolated from cell culture media using this procedure is approximately 90% pure and does not contain any free light chain, light-chain dimer, or contaminating serum albumin . In addition, murine IgG1 has been isolated from ascites fluid by direct application to the metal-affinity column . Murine IgG1 is recovered essentially free from albumin in approximately 60% purity, the principal contaminant being transferrin . This method facilitates further purification which is easily achieved by cation-exchange chromatography . The near complete removal of albumin makes metal-affinity chromatography an alternative to salt precipitation of antibody from ascites fluid . Recoveries of antibody from the column are high, typically greater than 90% . We have identified the location of the metal affinity in the IgG1 as being near the carboxy terminus of the heavy chain . A histidine-rich sequence is present in this region which offers the possibility of genetically engineering this sequence to form an even higher affinity metal binding site for potential application in antibody imaging and therapeutics. Vet Immunol Immunopathol, 1994 Oct, 43(1-3), 135 - 42 Development of immune responses in early pig ontogeny; Tlaskalova-Hogenova H et al.; Low amounts of immunoglobulins, produced without any known cause of stimulation, can be detected in sera and cells of fetal and colostrum deprived newborn pigs . These immunoglobulins are believed to represent the preimmune antibody repertoire on the basis of their polyspecificity and reactivity against self antigens . In vitro activation of liver and spleen cells with various polyclonal B cell activators (PBA) results in pronounced immunoglobulins synthesis as measured in the culture media by enzyme-linked immunosorbent assay . Intrauterine injection of fetal and germfree pigs with PBA led to increased IgM, IgG and IgA levels in sera . Specific responses during fetal development were studied after intrauterine immunization . Antibodies to the heapten and its carrier flagellin, could be detected 7 days after the immunization of 55-day-old fetuses . Fetal and colostrum germfree pigs may be useful experimental models in which developmental immunity can be studied in the absence of maternal antibodies and environmental antigens. Domest Anim Endocrinol, 1994 Oct, 11(4), 339 - 47 Insulin-like growth factor-II expression in developing skeletal muscle of double muscled and normal cattle; Gerrard DE et al.; Double muscled (DM) cattle possess nearly 40% more muscle fibers than normal muscled (NM) beef or dairy cattle . Previous work showed that serum from DM fetuses stimulated proliferation of L6 myoblasts to a greater extent than serum from NM fetuses . Although the exact role of insulin-like growth factor-II (IGF-II) in the regulation of fetal myogenesis is unknown, it has been shown to serve as a autocrine-acting growth factor during terminal differentiation of myoblasts in mitogen-depleted culture media . Delay of IGF-II expression may alter the ultimate number of muscle fibers formed during fetal development . To investigate this, forty-seven skeletal muscle and twenty-nine liver samples were collected from NM fetuses representing fetuses grouped by crown-rump lengths (CRL) of < 20, 21-30, 31-40, 41-50, 51-60, 61-70 or > 70 cm . Twelve DM fetuses representing 20, 40, 50, and 85 cm were compared to NM groups with the same CRL . Total RNA preparations from these samples were subjected to northern and dot blot analysis using rat IGF-II and beta actin cDNAs and a human 28S rRNA oligomer . IGF-II transcripts of 4.5, 3.6, 2.75, 2.5, and 1.15 kilobases (kb) were detected in liver and muscle RNA from both DM and NM fetuses . Liver IGF-II expression increased (P < 0.05) in both DM and NM fetuses with CRL . Mean concentrations of muscle IGF-II mRNA initially increased (P < 0.05), then decreased (P < 0.05) with CRL in DM and NM fetuses . Muscle IGF-II mRNA was greater (P < 0.05) for NM fetuses compared to DM fetuses at 20 cm CRL, whereas at 53 cm CRL, DM muscle IGF-II was greater (P < 0.05) than that of NM fetuses . These results show that the maximum expression of muscle IGF-II is delayed in DM fetuses compared to NM fetuses . This delayed expression may play an explicit role in controlling myogenesis in the development of double muscle cattle. J Neuroendocrinol, 1994 Oct, 6(5), 565 - 71 In situ hybridization detection of TSH beta subunit gene expression in the serum-free primary culture of the adult rat pituitary; Koibuchi N et al.; This study was designed to construct the primary culture system to detect the change in TSH beta subunit (TSH beta) gene expression in individual cells . Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight) . Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3, or 6 days with or without 1 nM triiodothyronine (T3) under the serum-free condition . In some cultures, TRH was added to a final concentration of 1 microM on 6, 12 or 24 h before fixation . Then the culture media were removed to measure TSH concentration . Cells were fixed with paraformaldehyde and hybridized with 35S-labeled RNA probe complementary to TSH beta mRNA . Emulsion autoradiography was subsequently performed . T3 treatment markedly suppressed relative cellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6 . TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSH beta mRNA on day 6 . TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6 . On day 2 and 3, although T3 treatment suppressed basal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T3 treatment . These results show that the thyroid hormone and TRH regulate TSH beta gene expression independently . Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells. J Biomater Appl, 1994 Oct, 9(2), 138 - 57 Studies on the standardization of cytotoxicity tests and new standard reference materials useful for evaluating the safety of biomaterials; Tsuchiya T; Standard reference materials (SRM) made of polyurethane films containing various amounts of cytotoxic compounds such as zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) are proposed . The cytotoxicity indices of five dithiocarbamates and the SRM films obtained by colony assay were compared with those by published standards, i.e., neutral red assay (NF S 90-702), agar diffusion assay (USP XXII), and millipore filter diffusion assay (DIN-V 13 930) . Among them, colony assay using 3 cell lines of Balb 3T3, L929 and V79 was found to be most sensitive . The extraction method and direct contact method were proposed as two test methods of colony assay . In the former, culture media after incubation with test materials were used as test solution, and in the latter, cells were directly cultured on test materials . The extraction method gave a linear relationship between the cytotoxic potentials of the SRM and its concentration of ZDEC or ZDBC . The cytotoxic potentials of the SRM correlated well with the thickness of inflammatory layer in rabbit muscle implantation tests of the SRM . The direct contact method was found to be useful to detect weak cytotoxicity because of its high sensitivity . Based on these data, we propose the colony assay and the two kinds of new SRMs as Japanese standard test method for evaluating the cytotoxicity of biomaterials. Zhonghua Nei Ke Za Zhi, 1994 Oct, 33(10), 663 - 5 {Pulmonary nocardia infection}; Cao WB et al.; Four cases of pulmonary Nocardia infection were reported and the 22 cases reported in our country between 1980 and 1993 were analyzed together for the purpose of revealing the clinical picture of this disease in China . There was approximately a ratio of two male patients to one female (18:8) with a mean age of 40 years in this series of patients . Various underlying diseases were found as predisposing factors in 69% (18/26) of the cases . 34.5% (9/26) of the cases had a history of corticosteroid therapy . Pleural effusion was identified in 50% (13/26) of the patients . 38% (10/26) of the patients died . Patients with primary pulmonary Nocardia infection had a better prognosis than those with secondary infection . Appropriate culture media, sufficient culture time and repeated cultures were recommended to improve the positive identification rate of this special causative agent . The sulfonamides were the drugs of first choice along with prompt drainage of thoracic empyema and abscess. Prostaglandins Leukot Essent Fatty Acids, 1994 Oct, 51(4), 257 - 62 The effect of Iloprost (ZK 36374) on isolated and transplanted pancreatic islet cells; Yegen C et al.; Several methods have been described for the prolongation of survival of isolated and transplanted islet cells . To investigate the effect of a stable prostacyclin analogue, ZK 36374 (Iloprost) on isolated and allotransplanted islet cell function, we studied 6 groups of rats: Group 1 (n = 7) animals underwent pancreatectomy and their islets were isolated and cultured by standard techniques . Group 2 (n = 8) animals were treated the same, except for the addition of Iloprost to the culture solutions . Group 3 (n = 7) animals were treated as group 1, but the isolated islets were transplanted to the subcapsular space of the left kidney of group 5 (n = 7) animals . Group 4 (n = 8) animals were treated as group 2, and the isolated islets were transplanted to group 6 (n = 8) animals . The insulin levels in the culture media obtained in group 1 and 2 were measured . In groups 5 and 6 blood glucose levels were measured and intraperitoneal glucose loading tests were performed . Histological examination was performed for both isolated and transplanted islets . The results showed that both insulin levels and histologic evaluation were better for group 2 than group 1 . Animals in group 6 reached normoglycemia on the fifth day following transplantation while it was the ninth day for group 5 . The intraperitoneal glucose loading test was tolerated better by group 6 animals . We conclude that Iloprost may be responsible for the improved results which seem to be due to its cytoprotective effect. J Neurosurg Anesthesiol, 1994 Oct, 6(4), 249 - 53 Propofol protects cultured rat hippocampal neurons against N-methyl-D-aspartate receptor-mediated glutamate toxicity; Hans P et al.; The effect of propofol on the toxicity induced by glutamate (GLU), N-methyl-D-aspartate (NMDA), kainate (KA), and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was investigated on cultured fetal rat hippocampal neurons . The degree of neuronal injury was quantified by measuring the release of the neuron-specific enolase (NSE) into the culture media . The toxicity induced by brief exposure to GLU (10(-4) M) or to NMDA (10(-4) M) was significantly reduced by propofol, whereas that elicited by KA, AMPA (10(-4) M), or long GLU exposure was unaffected . In conclusion, high concentrations of propofol significantly attenuate NMDA receptor-mediated glutamate neurotoxicity in vitro . Further studies are needed to confirm this beneficial effect in vivo and to evaluate propofol as a neuroprotective anesthetic agent in pathologies involving glutamate release and NMDA-mediated toxicity. Ann N Y Acad Sci, 1994 Sep 30, 734, 245 - 56 Interaction between steroid hormones and endometrial opioids; Gravanis A et al.; The opioids beta-endorphin and the dynorphins belong to two separate families of endogenous opioid peptides (EOP) . They are produced not only in the central nervous system but also in nonneural tissues where, as it appears, they act locally via paracrine mechanisms . These opioids have been shown to be produced at multiple sites along the mammalian reproductive tract including the intrauterine cavity . The aim of the present work was to find out if the well differentiated human endometrial cell line of Ishikawa, which has been shown to be a good in vitro model for the study of the effects of steroid hormones on human epithelial endometrium, expresses these two EOP . Northern blot hybridization of RNA from these cells showed the presence of a 1.2-kb POMC and a 2.4-kb PDYN transcript . Radioimmunoassay and gel filtration chromatography characterization of the immunoreactive (IR) opioid peptides present in the culture media showed the presence of IR-beta-endorphin and IR-dynorphins . The apparent molecular weight of IR-beta-endorphin was that of authentic beta-endorphin while the bulk of the IR-dynorphin had an apparent molecular weight of 8 kd . The secretion of both opioids could be increased by KCl-induced depolarization . Estrogen and glucocorticoids decreased, in a dose- and time-dependent manner, the secretion of beta-endorphin from the Ishikawa cells while progesterone and dihydrotestosterone did not have a statistically significant effect . The antiprogestin-antiglucocorticoid RU486 acted as an agonist, i.e., it diminished beta-endorphin secretion possibly via glucocorticoid receptors . On the other hand, the secretion of dynorphins was not affected by any of the steroids tested while LHRH, the inducer of gonadotropins and anterior pituitary dynorphins secretion, provoked a time- and dose-dependent increase of their secretion without affecting that of beta-endorphin . These data suggest that the regulation of endometrial opioids production is type-specific . Thus, it is possible that each type of endometrial opioid participates in different local homeostatic loops and exerts distinct paracrine effects. Cancer Lett, 1994 Sep 30, 85(1), 105 - 9 Differences between pulsatile or continuous exposure to melatonin on MCF-7 human breast cancer cell proliferation; Cos S et al.; We studied the different in vitro antiproliferative actions of melatonin on MCF-7 cells, depending on whether the cells are exposed to hormone concentrations which remain constant in culture media (Group I, 10(-9) M; Group II, 10(-11) M melatonin) or varying at 12 h intervals, thus simulating a diurnal rhythm: Group III, 12 h in 10(-9) M melatonin/12 h without melatonin (10(-9) M/0 12/12 h); Group IV, 10(-11) M/0 12/12 h; Group V, 10(-9) M/10(-11) M 12/12 h . After 5 days of culture, cell proliferation appeared significantly inhibited in Groups I and III, but not in Groups II and IV . However, the highest antiproliferative effect was obtained by sequential exposure to 10(-9) M/10(-11) M melatonin (Group V), which mimics the physiological rhythm of serum melatonin concentration. Biochem J, 1994 Sep 15, 302 ( Pt 3), 655 - 64 Iron requirement for cellular DNA damage and growth inhibition by hydrogen peroxide and bleomycin; Radtke K et al.; Studies with Euglena gracilis and HL-60 cells have assessed the need for intracellular iron in the mechanisms of inhibition of cell growth and DNA damage by H2O2 and bleomycin . Cell culture media were directly depleted of iron in order to deprive cells of nutrient iron . Major pools of cellular iron were reduced in both cell types . Nevertheless, iron bound in e.s.r.-observable haem protein and ribonucleotide diphosphate reductase in HL-60 cells was not decreased . In both control cell populations, there was a concentration-dependent reduction in proliferation and cell survival caused by H2O2 . In comparison, the proliferation rates of both iron-deficient cell types were significantly less sensitive to H2O2 . H2O2 caused concentration-dependent single-strand breakage in DNA in control HL-60 and Euglena gracilis cells . Iron deficiency reduced the amount of strand breaks in HL-60 cells at each concentration of H2O2 used . Single-strand breakage caused by H2O2 in Euglena gracilis was a direct function of the concentration of iron in which the cells had been grown . Growth inhibition and both single- and double-strand DNA damage caused by bleomycin were substantially reduced or eliminated in iron-deficient cells . Copper bleomycin behaved like metal-free bleomycin when assayed for the capacity to cause DNA damage in iron-normal and iron-deficient HL-60 cells . In contrast, iron bleomycin was equally active under the two conditions in these cells. Biochem J, 1994 Sep 1, 302 ( Pt 2), 451 - 4 Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11; Lafrance MH et al.; Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane . The ectodomain of NEP contains five putative N-glycosylation sites . In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme . Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition . Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form . The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme . Enzyme activity was decreased by deglycosylation . However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities . Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints . These constraints seem to be local for enzyme activity but more global for transport to the cell surface. Dev Biol, 1994 Sep, 165(1), 178 - 84 Cyclic AMP negatively modulates both Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein and membrane fusion of chick embryonic myoblasts; Baek HJ et al.; We have previously shown that Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein dramatically increases during the early period of myoblast fusion and treatment of calmodulin antagonists, such as trifluoperazine, blocks the fusion . Here, we show that cAMP treatment of primary cultures of chick embryonic myoblasts blocks 100-kDa protein phosphorylation . This effect is dose-dependent and can be reversed upon removal of the nucleotide from the culture media . However, cAMP shows little or no effect on accumulation of the 100-kDa protein . Furthermore, phosphorylation of the 100-kDa protein by the partially purified Ca2+/calmodulin-dependent protein kinase (CaM kinase III) from cAMP-treated cells occurs to a much lower extent than that from untreated cells . Nevertheless, cAMP-sensitive protein kinase does not seem to be directly involved in phosphorylation and inactivation of CaM kinase III, because preincubation of cAMP with the myoblast extracts lacking the endogenous 100-kDa protein does not show any effect on activity of CaM kinase III . Similar to its effect on 100-kDa protein phosphorylation, cAMP reversibly inhibits the fusion of cultured myoblasts . Moreover, treatment with forskolin or theophylline, which is known to elevate the intracellular cAMP level, also reversibly blocks both protein phosphorylation and myoblast fusion . On the other hand, cAMP shows little or no effect on accumulation of muscle-specific proteins, such as creatine kinase and tropomyosin . These results suggest that cAMP is involved in down-regulation of both 100-kDa protein phosphorylation and membrane fusion of cultured myoblasts . These results also suggest that the cAMP-mediated inhibition of 100-kDa protein phosphorylation may be associated with its inhibitory effect on myoblast fusion. Exp Cell Res, 1994 Sep, 214(1), 145 - 53 Fatty acid modulation of epidermal growth factor-induced mouse mammary epithelial cell proliferation in vitro; Sylvester PW et al.; Mammary epithelial cells were isolated from midpregnant BALB/c mice, grown in primary culture within collagen gels, and maintained with serum-free medium containing 10 ng/ml epidermal growth factor (EGF) as the mitogen . Supplementation of culture medium with the saturated fatty acid, Na-stearate (18:0), significantly attenuated, whereas treatment with the unsaturated fatty acid, Na-arachidonate (20:4), significantly enhanced mammary epithelial cell proliferation, as compared to untreated controls . Treatment with various doses of either 18:0 or 20:4 was also found to result in a direct dose-dependent enrichment of mammary epithelial cell membrane fatty acid composition and a concurrent decrease in the relative levels of other membrane fatty acids, as determined by gas chromatography . Administration of the prostaglandin synthesis inhibitor, indomethacin, significantly inhibited EGF-induced cell growth in all treatment groups, but did not alter the relative inhibitory (18:0) or stimulatory (20:4) effects of fatty acid treatment . EGF-induced PKC translocation into the membrane fraction of mammary epithelial cells was enhanced in 20:4 and attenuated in 18:0 treatment groups, as compared to controls . Western blot analysis of phospholipid-dependent protein kinase C isoenzymes showed that PKC alpha was the predominant isoenzyme present in mouse mammary epithelial cells grown in primary culture, and the molecular weight of this PKC isoenzyme was determined to be 85 kDa . These results suggest that supplementation of culture media with specific fatty acids is associated with significant alterations in mammary epithelial cell membrane fatty acid composition, PKC activation, and mitogenic responsiveness . Since EGF can induce both PKC activation and cell proliferation, and because PKC activation requires membrane-derived phospholipids and diacylglycerol, these data suggest that specific fatty acid modulation of mammary epithelial cell mitogenesis is mediated through alterations in PKC alpha activation. J Gen Virol, 1994 Sep, 75 ( Pt 9), 2213 - 21 Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus; Hertig C et al.; Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rVVs) expressing regions of the bovine leukaemia virus genome . Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rVV carrying the gag and truncated protease (pro) gene . Processing of Pr44 was observed after infection of cells with a rVV carrying the gag and pro gene or a rVV expressing the gag, pro and polymerase (pol) gene . Reverse transcriptase activity was detected only in association with particles produced by gag-, pro- and pol-expressing recombinants . Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane . Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rVVs expressing gag and pro or gag, pro and pol formed mature particles. Diabetes, 1994 Sep, 43(9), 1138 - 45 Effects of glucose on insulin secretion, glucokinase activity, and transgene expression in transgenic mouse islets containing an upstream glucokinase promoter-human growth hormone fusion gene; Liang Y et al.; We have analyzed in organ culture the effects of glucose on glucose-induced insulin secretion, glucokinase (GK) activity, and human growth hormone (hGH) expression in pancreatic islets from transgenic mice containing an upstream GK promoter-hGH fusion gene . Freshly isolated islets from these mice had a normal insulin secretory response to glucose but showed subtle defects after culture in low or high glucose for 4 days that may have been due to the accumulation of hGH in the culture media . Islets cultured from both normal and transgenic mice had approximately a fourfold induction of GK activity in response to an increased concentration of glucose in the culture media, whereas no such change in total islet hGH production was observed . Immunocytochemical localization of hGH in islets cultured in 3 mM glucose showed a pattern similar to that in freshly isolated islets . However, after culture in 30 mM glucose, hGH immunostaining became strikingly more heterogeneous . We conclude 1) that GK-hGH transgene expression does not appear to adversely affect glucose-stimulated insulin secretion in vivo or in freshly isolated islets, 2) that glucose does not induce transgene expression, thus providing additional evidence against an effect of glucose on GK gene transcription in the islet, and 3) that glucose stimulates the co-release of hGH with insulin, thereby enhancing the heterogeneous staining pattern seen among pancreatic beta-cells. Cell Immunol, 1994 Sep, 157(2), 549 - 55 Interleukin-1 and interferon-alpha augment interleukin-2 (IL-2) production by distinct mechanisms at the IL-2 mRNA level; Holan V et al.; The production of interleukin-2 (IL-2) by phytohemaglutinin (PHA)-stimulated cells of human leukemia T cell line MOLT-16 can be significantly increased by interleukin-1 (IL-1) or interferon-alpha (IFN-alpha) . The enhancing effect of IL-1 and IFN-alpha on IL-2 production was studied at the IL-2 mRNA level . We show that IL-1 enhances considerably and transiently, with the maximum level between 1 and 2 hr after stimulation, the expression of IL-2 mRNA in the PHA-stimulated cells . The level of IL-2 mRNA declined rapidly within 4 to 6 hr after stimulation in both PHA- and PHA plus IL-1-stimulated cell cultures . On the contrary, IFN-alpha does not elevate the level of IL-2 mRNA above the level in PHA-stimulated cultures, but maintains an enhanced level of IL-2 mRNA in the activated cells for more than 6 hr after stimulation . These observations correlate well with the kinetics of IL-2 protein production into the culture media . The results thus suggest that IL-1 and IFN-alpha may exert an enhancing effect on IL-2 production by distinct mechanisms . In addition, none of the five other lymphokines tested (i.e., IL-2, IL-3, IL-4, IL-5, and IL-6) had any significant effect on IL-2 mRNA expression in the activated MOLT-16 cells. Cell Biol Int, 1994 Sep, 18(9), 897 - 901 Effect of extracellular K+ on hatching and outgrowth of mouse blastocysts in vitro; Jin Z et al.; The dependence of blastocyst development on the extracellular Na+/K+ ratio was investigated in an in vitro system . Hatching and outgrowth of mouse blastocysts was enhanced at Na+/K+ ratios between 3 and 10 compared to the ratio of about 25 typical for most culture media and serum . At a Na+/K+ ratio of 2, blastocyst hatching and outgrowth were inhibited . The requirement of blastocyst development for relatively high extracellular K+ concentrations agrees with the fact that K+ concentrations in oviduct and uterine secretions are higher than in serum . The findings can also be relevant in optimizing in vitro culturing techniques for blastocysts. J Orthop Res, 1994 Sep, 12(5), 720 - 31 Response of three murine macrophage populations to particulate debris: bone resorption in organ cultures; Glant TT et al.; Particulate wear debris from bone cement or prosthetic components can stimulate macrophages to cause bone resorption . We compared the effect of particle composition (titanium and polymethylmethacrylate as inherent components of prosthetic materials or bone cement and polystyrene as a reference material) on the secretion of interleukin-1 and prostaglandin E2 by peritoneal macrophages and monocyte/macrophage cell lines (P388D1 and IC-21) and on the bone-resorbing activity of conditioned medium harvested from these particle-challenged macrophages . Titanium particles (1-3 microns) in peritoneal macrophage cultures exhibited significantly enhanced bone-resorbing activity measured as 45Ca release, whereas polymethylmethacrylate and polystyrene exhibited this effect to a greater extent in the P388D1 and IC-21 monocyte/macrophage cultures . Although exogenous prostaglandin E2 and recombinant human interleukin-1 could significantly increase the 45Ca release and indomethacin significantly reduced both the spontaneous calcium efflux and active 45Ca release from calvarial bones labeled in vivo, the levels of interleukin-1 and prostaglandin E2, a