Culture medium

Arch Microbiol, 1994, 161(2), 152 - 5
Morphological and molecular characterization of Frankia sp . isolates from nodules of Alnus nepalensis Don; Ganesh G et al.; Nodules collected from Alnus nepalensis growing in mixed forest stands at three different sites around Shillong, were crushed in various culture media to obtain isolates of Frankia . The isolates were found to have typical Frankia morphology as revealed by the scanning electron microscope . Seedlings inoculated with isolates or crushed nodules formed nitrogen fixing nodules . Frankia specific DNA probes amplified the DNA of the tested isolate AnpUS4 . Partial nucleotide sequence of the 16S rRNA gene indicated that AnpUS4 was phylogenetically distinct from all other Frankia strains characterized so far.

J Endocrinol, 1994 Jan, 140(1), 125 - 35
Expression of growth hormone-binding protein with a hydrophilic carboxyl terminus by the mouse placenta: studies in vivo and in vitro; Barnard R et al.; GH-binding protein (GHBP) or GH receptor is present in numerous extrahepatic tissues in the rodent . From mid- to late gestation in the mouse, the maternal serum concentration of GHBP increases 30- to 50-fold . We have investigated whether the placenta might synthesize GHBP and potentially contribute to this increase . RNA was isolated from placentas and subjected to Northern analysis using a cDNA probe to the shared region of GHBP and GH receptor-encoding mRNAs . From day 8 to day 18 of gestation, the GHBP-encoding mRNA (1.4 kb) increased 45-fold in quantity . The GH receptor-encoding mRNA (4.2 kb) increased sixfold by day 14 and then remained steady until day 18 . These changes which were not co-ordinated parallel reported changes in the steady-state concentrations of 1.4 and 4.2 kb mRNAs in maternal liver, suggesting shared regulatory factors . Extracts of freshly isolated trophoblasts were assayed for GHBP with a radioimmunoassay specific for GHBP with a hydrophilic carboxyl terminus . The cytosolic content of immunoreactive GHBP increased fourfold from mid- to late gestation . Trophoblasts were isolated from placentas and cultured for 2 days on collagen gels in defined medium . Cultured cells were at least 90% viable and secreted mouse placental lactogen-II (mPL-II) . Immunocytochemistry was carried out simultaneously on cells cultured from day 7 to day 17 of gestation using a monoclonal antibody (MAb 4.3), which recognizes the hydrophilic C-terminus of GHBP . Cell-localized GHBP was present in trophoblasts cultured for 2 days, but GHBP was not detectable by radioimmunoassay or by immunoprecipitation in concentrated culture media from cultures treated with 100 ng mouse GH/ml or 100 ng mPL-II/ml or from untreated cultures . RNA was isolated from cells cultured in an identical manner to those analysed by immunocytochemistry . Three GH receptor/GHBP mRNA species of 8, 4.2 and 1.4 kb were observed . The quantity of 4.2 and 1.4 kb mRNAs did not change significantly in cultures from day 7 to day 15 of gestation but, in cultures from day 17 of gestation, the amount of 1.4 kb mRNA dropped significantly, while that of the 4.2 kb mRNA remained unchanged . GHBP- and GH receptor-encoding mRNAs are not co-ordinately regulated in vivo or in vitro . Although mPL-II was secreted into the medium by cultured trophoblasts, secretion of GHBP could not be detected . The culture medium may not contain the specific factors required for secretion of placental GHBP, or placental GHBP may not be destined for secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Br J Cancer, 1994 Jan, 69(1), 125 - 9
Constitutive production of multiple colony-stimulating factors in patients with lung cancer associated with neutrophilia; Adachi N et al.; Production of colony-stimulating factor (CSF) was examined in three patients with lung cancer associated with neutrophilia . All three patients presented a marked increase in neutrophil count (26,000-39,000 microliters-1) that continued at least for 3 weeks and rapidly disappeared after surgical removal of the tumours . Culture media (CM) incubated with the excised tumour tissues stimulated the colony formation of bone marrow myeloid progenitor cells in vitro . Northern blot analysis of poly(A)+ RNA from the tumour tissues revealed a constitutive expression of granulocyte (G), macrophage (M), and granulocyte-macrophage (GM) CSF genes in all tumours . Immunoassay specific for these CSFs revealed that G- and M-CSF immunoreactivity was detected in all CM and GM-CSF protein in two out of three CM . The plasma CSF levels also increased before operation and decreased to normal or near-normal range after operation . In contrast, tumour cell CM obtained from two lung cancer patients without leucocytosis neither stimulated haematopoietic colony formation nor contained immunoreactive CSFs . These results indicated that the neutrophilia found in the three patients was probably caused by constitutive production of multiple CSFs by lung cancer cells.

J Cancer Res Clin Oncol, 1994, 120(3), 137 - 42
Production of insulin-like growth factor binding proteins by human ovarian carcinoma cells; Hofmann J et al.; Cells of the human ovarian carcinoma lines EFO-21, EFO-27, MFO-35 and MFO-36 secrete binding proteins for insulin-like growth factors (IGFBPs) into their culture media . By sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting, seven groups of IGFBPs with molecular masses of 25, 30 (doublet), 34, 37, 40, 45, and 50 kDa were observed, depending on the cell line under investigation . By Northern blot analyses using cDNAs or oligonucleotides specific for the six types of IGFBP (IGFBP-1 to IGFBP-6), mRNA for all IGFBPs tested except for IGFBP-1 could be detected in the ovarian carcinoma cell extracts . In detail, analysis of EFO-21 protein products by SDS-PAGE yielded IGFBPs of 25, 34, and 50 kDa; extracts of EFO-21 cells contained mRNAs for IGFBP-2, -3, -4, and -6 . EFO-27 cells produced IGFBPs of 40 kDa and 45 kDa as determined by SDS-PAGE, and mRNAs for IGFBP-3, -4, and -6 were detected . In the conditioned medium of MFO-35 cells, IGFBPs of 25, 30 (doublet), 34, 37, 40, and 45 kDa were observed by SDS-PAGE, while mRNAs for the five proteins IGFBP-2 to IGFBP-6 were found . MFO-36 cells produced IGFBPs of 34 kDa and 50 kDa as determined by SDS-PAGE, and the cells expressed mRNAs for IGFBP-2, -3, -4, and -6 . In relation to published molecular mass data of the known IGFBPs, the size of the secreted proteins could be correlated to the mRNA patterns expressed by the ovarian carcinoma cells . It is concluded that ovarian carcinoma cells frequently express IGFBP-3, -4, and -6 and, to a lesser extent, IGFBP-2; the expression of IGFBP-5 appears as a rather rare event, while IGFBP-1 was not found to be expressed in ovarian carcinoma cells.

Neurosci Lett, 1993 Dec 24, 164(1-2), 141 - 4
Neurotrophic effects of substance P on hippocampal neurons in vitro; Whitty CJ et al.; The potential neurotrophic effect of substance P-like immunoreactivity present in culture media was assessed in rat embryonic day 18 hippocampal cultures . The neurokinin-1 (substance P) receptor antagonist CP-96345 induced neurotoxicity that was dose dependent and attenuated by addition of substance P or the neurokinin-1 agonist {Sar9,Met(O2)11}-SP . These studies suggest that under some conditions neurokinin-1 receptor stimulation promotes neuronal survival.

Cancer Res, 1993 Dec 15, 53(24), 5970 - 6
In vitro cytotoxicity, protein binding, red blood cell partitioning, and biotransformation of oxaliplatin; Pendyala L et al.; The in vitro cytotoxicity, protein binding, partitioning of platinum from whole blood into erythrocytes, its exchange back into plasma, and the in vitro biotransformation in plasma were studied for the new nonnephrotoxic platinum analogue oxaliplatin . The cytotoxicity studies were carried out against a panel of human tumor cell lines derived from carcinomas of the ovary (A2780, A2780/cp), bladder (TCCSUP, RT4), colon (HT-29), melanoma (SKMEL-2, HTB144), and glioma (U373MG and U87MG) . The relative potency of the five platinum complexes was oxaliplatin = tetraplatin > cisplatin > iproplatin > carboplatin . Oxaliplatin was active against HT-29 and only minimally cross-resistant with cisplatin against A2780/cp . Both bladder carcinoma cell lines, both melanoma cell lines, and one of the two glioblastoma cell lines were resistant to both oxaliplatin and tetraplatin . The cytotoxicity profiles of the drug pairs oxaliplatin-tetraplatin and cisplatin-carboplatin showed statistically significant correlation by the Spearman rank correlation test . Oxaliplatin was similar to cisplatin and tetraplatin in protein binding; 85-88% of all platinum from oxaliplatin (5, 10, or 20 micrograms/ml) was bound to plasma proteins within the first 5 h with an average half-life of 1.71 +/- 0.06 h . When oxaliplatin was incubated in whole blood (5, 10, and 20 micrograms/ml), the erythrocytes took up 37.1 +/- 2.1% of the total platinum in 2 h (maximum uptake) which was not exchangeable into plasma . Thus the erythrocyte-bound fraction does not serve as a reservoir of drug . In plasma, oxaliplatin was unchanged at 0.5 h, but at 1 h, 30% of the total platinum in plasma was in a peak which had identical retention to that of (trans-1,2-diaminocyclohexane)dichloroplatinum(II), the major biotransformation product of tetraplatin . At 2 h, (trans-1,2-diaminocyclohexane)dichloroplatinum(II) and three other platinum-containing peaks were detected but no unchanged oxaliplatin . All the platinum eluted in a single peak near the solvent front at 4 h . The marked similarity in cytotoxicity between oxaliplatin and tetraplatin may be due to the formation of (trans-1,2-diaminocyclohexane)dichloroplatinum(II) in tissue culture media.

J Virol Methods, 1993 Dec 15, 45(2), 219 - 28
Cytofluorographic analysis of effects of interferons on expression of human cytomegalovirus proteins; Torigoe S et al.; The appearance of cytomegalovirus (CMV) proteins in infected fibroblasts was determined by flow cytometry . The sequential production of immediate early (IE), early (E), and late (L) proteins reacting with respective monoclonal antibodies (mAbs) E13, 58/5, and 24/4 was determined in fibroblasts infected with the AD-169 strain of CMV . The percentage of cells expressing CMV proteins and the intensity of fluorescence within cells were determined from day 1 to day 7 post-infection . The effect of interferons (IFNs) alpha, beta, gamma on expression of CMV proteins was analyzed using flow cytometry . IFNs inhibited E and L protein production at days 3 and 6 post-infection in a dose-dependent manner . This inhibitory effect on protein expression was associated with a reduction in release of infectious CMV into culture media . The method described here for detection of CMV proteins using flow cytometry may be useful for basic studies of gene expression and for diagnostic purposes.

Ann Plast Surg, 1993 Dec, 31(6), 499 - 503
Effect of skin flap ischemia on plasma endothelin-1 levels; Matsuzaki K; A number of studies have been done relating to vasospasm . Vasospasm within the microvasculature of a flap can be one of the causes of ischemia and nonviability . Endothelin-1 (ET-1), a 21-amino acid polypeptide isolated from vascular endothelium culture media, is reported to be one of the most potent vasoconstrictors known . This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia influenced plasma ET-1 levels . After the ischemic insult, blood was drawn from the venous effluent of the flaps . Plasma ET-1 levels after 6 hours of ischemia were significantly increased compared with nonischemic controls; they were 29 pM, i.e., almost enough to induce vasoconstriction of arterioles . These results suggest that ET-1 is one of the factors responsible for partial necrosis of the skin flap, which contributes to the genesis of the no-reflow phenomenon.

Biol Reprod, 1993 Dec, 49(6), 1288 - 92
Effects of glucose and fructose on fertilization, cleavage, and viability of mouse embryos in vitro; Sakkas D et al.; In this study we examined the role of glucose, and the use of fructose as a replacement, in embryo culture medium . Three embryo culture media were used: a routine embryo culture medium (M16), M16 without glucose (M16-G) and M16-G supplemented with fructose (M16F-G) . Their effect on fertilization, rate of cleavage, and embryo viability were examined in both an outbred (OF1) and an inbred (C57Bl) mouse strain . Of the three media, only M16 was found to support fertilization . In vitro-fertilized embryos from OF1 oocytes and C57Bl oocytes and OF1 sperm were placed in the different media at the early 2-cell stage . In 65% of OF1 embryos cultured in M16, development was blocked at the 2-cell stage, whereas in M16-G and M16F-G embryos, only 22% and 32%, respectively, were blocked . M16F-G medium also produced morulae and blastocysts with higher cell numbers than M16-G . In vitro-fertilized C57Bl 2-cell embryos cultured in M16 displayed retarded cleavage to the 4-cell stage compared to embryos cultured in M16-G and M16F-G . In contrast, the morulae and blastocyst cell numbers were significantly lower in M16-G compared to M16 and M16F-G . The viability of morulae and blastocysts obtained from OF1 and C57Bl embryos cultured in M16-G and M16F-G was lower compared to control C57Bl morulae and blastocysts cultured in M16 . The results show that although morphologically normal embryos could be obtained in M16-G and M16F-G, inherent anomalies existed that limited viability.

Br J Dermatol, 1993 Dec, 129(6), 678 - 88
A calmodulin-like protein as an extracellular mitogen for the keratinocyte; Goberdhan NJ et al.; This study investigated the importance of extracellular calmodulin to the proliferation of the keratinocyte . Normal keratinocytes in culture produced a calmodulin-like protein in their culture media, the level of which increased abruptly and transiently during their growth . This protein was calmodulin-like, in that it specifically bound to a calmodulin affinity column, exhibited calmodulin-like immunoreactivity in both an ELISA and on immunoblots when immunostained with a monoclonal antibody against calmodulin, had an apparent M(r) between 18,000 and 20,000, and stimulated activity in a calmodulin-dependent phosphodiesterase enzyme assay . Addition of exogenous pure calmodulin was of no further mitogenic benefit to the keratinocytes, and slightly reduced proliferation under the culture conditions used . However, addition of either a neutralizing antibody to calmodulin, or W7-agarose, to the culture media of proliferating cells markedly inhibited their proliferation . Accordingly, a calmodulin-like protein was found to satisfy all but one of the criteria for its action as an autocrine growth factor for the keratinocyte . We propose that the lack of mitogenic response to calmodulin in vitro is due to the cell meeting its own requirement for extracellular calmodulin.

J Parasitol, 1993 Dec, 79(6), 913 - 21
Culture of cells from juvenile worms of Schistosoma mansoni; Hobbs DJ et al.; Tissue disruption methods were developed and serum-free cell culture media formulated for the maintenance in vitro of cells from juvenile worms (day 18 after infection) of Schistosoma mansoni . Cultures maintained viability for up to 6 mo when plated on a feeder layer of irradiated rat liver cells and survived primarily as clusters of small (2.5-4 microns diameter) cells with a high nuclear-to-cytoplasmic ratio and relatively few organelles identified by electron microscopy . Cultures synthesized a protein profile similar to that of intact worms, and the cell clusters maintained a time- and concentration-dependent contractile response to serotonin . Cells synthesizing DNA were detected by precursor incorporation and flow cytometry in cultures initially and also after several weeks in vitro, although the percentage of cells synthesizing DNA decreased with time . Efforts to identify peptide growth factor-responsive tyrosine phosphorylation were negative, and the overall amount of S . mansoni phosphotyrosine-containing proteins identified by western blot with anti-phosphotyrosine monoclonal antibody was much less than that found in a peptide growth factor-responsive mouse cell line.

J Cell Physiol, 1993 Dec, 157(3), 594 - 602
Lipophilic impurity of phenol red is a potent cation transport modulator; Hopp L et al.; Previously, we described substantial alterations in the Na+ and K+ homeostasis of human skin fibroblasts following removal of fetal bovine serum (FBS) . Herein, we report that FBS removal per se does not cause any cellular ionic changes unless a lipophilic impurity of commercial phenol red preparations is present . This substance accelerates 86Rb+ efflux four to seven times, causes a four to eight time increase in cellular Na+, and a 40-70% reduction in cellular K+ contents . FBS (10%) or albumin (0.8%) appears to bind the impurity thus inhibiting its action . The increased cellular Na+ and decreased K+ contents do not return to baseline within 4 hours following the removal of the phenol red extract . However, albumin completely reverses the cellular cationic changes that develop during a 2 hour exposure of the cells to the free substance . The reversibility of its action by albumin suggests that the substance exerts its effect on or within the cell membrane and not intracellularly . Among seven different cell lines tested the 86Rb+ efflux from, and the Na(+)-K+ contents of, COS-7 and Hs68 cells also responded to unpurified phenol red in a way similar to human fibroblasts . The amount of the phenol red contaminant is manufacturer dependent . As little as 0.5 microM phenol red, from one vendor, was sufficient to elicit response in the 86Rb+ efflux . Given that the impurity is unlikely to be more than a small fraction of phenol red, it seems to be a potent ionic transport modulator . Based on these results, the presence of commercial phenol red in serum-free growth or test media, including the increasing variety of chemically defined culture media, should be considered as a potential confounding factor in measurements that depend on intracellular Na(+)-K+ homeostasis . The findings of such earlier studies may need to be reconsidered if the cells were exposed to unbound phenol red . We recommend that, until the manufacturers further refine their product, phenol red be purified by ether extraction before its use . The evaluation of the potential physiologic or pharmacologic relevance of this potent cation transport modulator awaits its isolation.

Hepatology, 1993 Dec, 18(6), 1465 - 76
Degradation and intracellular accumulation of a residualizing hyaluronan derivative by liver endothelial cells; McGary CT et al.; The release and intracellular accumulation of 125I-hyaluronan degradation products was studied in cultured liver endothelial cells with hyaluronan oligosaccharides (relative molecular mass = approximately 44,000) uniquely modified and radiolabeled at the terminal reducing sugar . Two methods were combined to measure 125I-hyaluronan degradation by liver endothelial cells . (a) Cetylpyridinium chloride precipitation of hyaluronan oligosaccharides was used as a rapid, convenient assay to monitor the appearance of hyaluronan degradation products . Hyaluronan oligosaccharides less than 54 to 60 monosaccharides in length were not precipitated with cetylpyridinium chloride and thus were assessed as degraded . (b) Gel filtration chromatography was used to estimate the size range of oligosaccharides produced by liver endothelial cells . After internalization of 125I-hyaluronan, liver endothelial cells released radioactive degradation products into the culture media after a lag period of 2.5 to 3.0 hr . The intracellular accumulation of degraded 125I-hyaluronan was linear for at least 2 hr even though no degradation products were released . The long lag before release of degraded 125I-hyaluronan is likely caused by the modified chemical structure at the reducing end of the hyaluronan derivative; the derivative acts like a residualizing label . After this lag the release of degraded 125I-hyaluronan proceeded linearly for up to 12 hr . The extracellular 125I-hyaluronan degradation products eluted with a distribution coefficient of 1.3 on a gel filtration column . The major intracellular 125I-labeled degradation product showed the same retardation (distribution coefficient = 1.3) . This retention may be caused by the hydrophobic aromatic and alkyl modifications to the former reducing sugar, also characteristics of a residualizing label . In addition, at least two larger minor intermediates were observed intracellularly . The rate of intracellular 125I-hyaluronan degradation was dependent on hyaluronan concentration and reached a maximal rate (159 molecules/cell/sec) at 2 x 10(-7) mol/L . This was about half the maximal rate of endocytosis (285 molecules/cell/sec) at a hyaluronan concentration of 1.3 x 10(-7) mol/L . The apparent ligand concentration that gives half-maximal responses for endocytosis and intracellular degradation was 0.6 x 10(-7) and 1.0 x 10(-7) mol/L, respectively.

Endocrinology, 1993 Dec, 133(6), 2568 - 73
Expression, regulation, and production of tumor necrosis factor-alpha in mouse testicular interstitial macrophages in vitro; Xiong Y et al.; Tumor necrosis factor-alpha (TNF alpha) is a cytokine principally secreted from macrophages and monocytes activated by agents such as lipopolysaccharide (LPS) . We have recently shown that TNF alpha inhibited mouse Leydig cell steroidogenesis in vitro . LPS injection has also been shown to repress Leydig cell function and induce TNF alpha messenger RNA (mRNA) expression in testicular interstitial macrophages in vivo . A paracrine regulation of Leydig cell testosterone synthesis by testicular interstitial macrophages via TNF alpha has been proposed . To further support this possibility, we examined whether LPS can induce TNF alpha mRNA expression and protein production in testicular interstitial macrophages in vitro . The regulation of LPS-stimulated TNF alpha mRNA expression in vitro was also investigated by employing the protein synthesis inhibitor cycloheximide (CHX) . TNF alpha secretion into culture supernatants was examined by both bioassay and enzyme-linked immunosorbent assay . Isolated testicular interstitial macrophages were cultured for 24 h before the initiation of treatments . Cells were treated with or without LPS (1.0 micrograms/ml) and in the presence or absence of CHX (5.0 micrograms/ml) at different time points . Northern blot analysis showed that TNF alpha mRNA was rapidly and significantly induced by LPS in testicular interstitial macrophages . The peak expression was at 2 h after the treatment, which was 8.3 +/- 2.6-fold over the control (P < 0.05) . TNF alpha mRNA then declined quickly and completely disappeared by 8 h after LPS treatment . In contrast to this rapid and transient induction of TNF alpha message by LPS alone, CHX extended the induction and caused a marked increase in LPS-induced TNF alpha mRNA at 2 and 6 h . CHX induced more LPS-stimulated TNF alpha mRNA at 6 h than that at 2 h . At 3 h after LPS treatment, TNF alpha secretion was significantly stimulated (5.6 +/- 1.2 U/micrograms macrophage DNA) measured by L929 tumor fibroblast cytotoxicity . TNF alpha was also detected by enzyme-linked immunosorbent assay in culture media of testicular interstitial macrophages treated with control medium or LPS for 1, 2, and 6 h . TNF alpha secretion was increased in a time-dependent way . There are significantly higher LPS-induced TNF alpha levels in culture media at 2 h (35.4 +/- 2.2 pg/micrograms macrophage DNA) and 6 h (85.5 +/- 11.1 pg/micrograms macrophage DNA) than those in control groups . The current study demonstrates that LPS activates testicular interstitial macrophages to express TNF alpha mRNA and secrete TNF alpha protein in vitro.

Exp Eye Res, 1993 Dec, 57(6), 747 - 51
DL-propranolol inhibits lens hexokinase activity and affects lens optics; Dovrat A et al.; A clinico-biochemical study indicated that the beta-blocker DL-propranolol may affect human lens epithelial hexokinase (HK) activity . In that study five key enzymes were analysed in 192 freshly excised human lens epithelia obtained during cataract surgery . In a large number of patients the epithelial HK was found to be inactive . Medical records of these patients showed widespread use of the drug DL-propranolol . In vitro experiments demonstrated a direct inhibitory effect of the drug on human lens HK activity . Lens refractive function was monitored during long term bovine lens culture experiments in which the potential cataractogenic agent was added to the culture media . DL-propranolol in a concentration of 0.1 mM reduced HK activity in bovine lens epithelium after 72 hr in organ culture and disrupted lens light focusing ability after 250 hr of incubation . Kinetic studies of HK inhibition suggested a competitive inhibitory effect of the drug on the enzyme.

Int J Biochem, 1993 Dec, 25(12), 1865 - 71
Comparative structural analysis of human and rat 65 kDa tumor-associated phosphoproteins; Mirowski M et al.; 1 . A 65 kDa-tumor-associated protein (p65) was isolated from human and rat carcinoma cell culture media . Antibodies raised to the rat protein recognized an antigenically related protein in human cancer cell line . 2 . Amino acid composition, N-terminal and internal sequence as well as peptide map and western blot analysis of the p65 strongly suggest a high degree of homology between the human and rat p65 proteins . 3 . Homology searches indicated that p65 was not homologous to previously sequenced proteins, but that it may be related to proteins of the steroid receptor superfamily of genes, especially c-erb A gene.

Anal Biochem, 1993 Dec, 215(2), 171 - 9
An assay for matrix metalloproteinases and other proteases acting on proteoglycans, casein, or gelatin; Manicourt DH et al.; We have set up a quantitative and sensitive enzymatic assay for proteases of different classes acting on proteoglycans, casein, or gelatin . Radiolabeled substrates were covalently attached to insoluble microcarriers and assays were performed in 96-well plates . Protease activities were determined by the release of labeled degradation products . Time- and dose-response curves were linear when the solubilization of labeled substrates did not exceed 15-20% of the initially bound molecules . Results were compared to those from zymographic analyses on proteoglycan-, gelatin-, and casein-polyacrylamide gels, as well as to the results obtained with conventional assays using soluble {3H}-casein and {3H}gelatin . Our assay procedure was more sensitive than other available methods: it detected picogram amounts of trypsin as well as picogram or nanogram amounts of the purified human matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9, depending on the specific activities of these MMPs on the different substrates . Our new procedure was appropriate for assaying the MMPs present in crude culture media conditioned by chondrocytes cultivated under various conditions.

Nihon Kyobu Shikkan Gakkai Zasshi, 1993 Dec, 31(12), 1534 - 41
{Production and inhibition of eosinophil colony stimulating factor by lymphocytes from patients with bronchial asthma}; Tanida K et al.; To clarify the mechanism of the eosinophilia in patients with bronchial asthma (BA), we examined the production and inhibition of eosinophil colony stimulating factor (Eo-CSF) of mononuclear cells (MNC) and lymphocytes from BA . After bone marrow cells from normal volunteers (NA) were incubated with culture media of MNC or lymphocytes cultured in the presence of interleukin-2 (IL-2, 1 U/ml) or Dermatophagoides farinae (10 micrograms/ml, Df), the number of eosinophils was increased and GM-CSF production was increased after 5 days of culture . Interleukin 5 (IL-5) production was increased in some cases of BA, but did not differ between BA and NA delete . When MNC were incubated in the presence of IL-2 and interleukin 4 (IL-4), neither eosinophilia nor GM-CSF production occurred . These results suggest first that MNC and T cells in BA have increased responsiveness to IL-2 and Df, second that increased GM-CSF production partly causes eosinophilia, and third that IL-4 inhibited IL-2-induced eosinophil proliferation by inhibition of GM-CSF production.

J Bone Miner Res, 1993 Dec, 8 Suppl 2, S505 - 10
Cytokines and growth factors in the regulation of bone remodeling; Mundy GR; Osteoporosis and periodontal disease both represent examples of abnormal bone remodeling . As knowledge of the cellular and molecular events in the normal bone remodeling process has accumulated in the last decade, better understanding of the pathophysiology of bone loss associated with periodontal disease and with aging has occurred . This short review does not attempt to include all aspects of this topic but covers specific areas in which there have been recent advances . (1) Observations made in the last few years have indicated that a hierarchy of both receptor and nonreceptor tyrosine kinases may be involved in normal osteoclastic bone resorption and that certain members of these tyrosine kinase families may mediate cytokine effects . Studies in the op/op variant of murine osteopetrosis have shown that normal production of monocyte-macrophage colony-stimulating factor 1 (M-CSF, also called CSF-1) and activation of its receptor (the receptor tyrosine kinase c-fms) are required for normal osteoclast formation . (2) Studies in mice made deficient in nonreceptor tyrosine kinase by gene knockout have shown that expression of this nonreceptor tyrosine kinase is required for normal osteoclast action and ruffled border formation, although not for osteoclast formation . (3) Recent studies have shown that in addition to prostaglandins of the E series, other arachidonic acid metabolites may be involved in normal and abnormal osteoclastic bone resorption . 5-Lipoxygenase metabolites, the leukotrienes, stimulate isolated osteoclasts to form resorption pits as well as cause osteoclastic bone resorption in organ cultures of neonatal mouse calvariae . These compounds, which are unstable in tissue culture media, are readily inhibitable by agents that inhibit 5-lipoxygenase enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Parasitology, 1993 Dec, 107 ( Pt 5), 559 - 66
External stimuli and intracellular signalling in the modification of the nematode surface during transition to the mammalian host environment; Proudfoot L et al.; Previous work has shown that the surface of infective larvae of parasitic nematodes will not bind the fluorescent lipid analogue 5-N-(octadecanoyl)aminofluorescein (AF18) until after exposure of the parasite to mammalian tissue-culture conditions . In this study, culture media which are permissive or non-permissive for the acquisition of lipophilicity for AF18 were altered in order to examine possible stimuli involved . This showed that external alkaline pH and high sodium ion concentration were highly stimulatory . The internal signalling pathways which may be involved in the surface alteration were then examined using agents which are known to affect intracellular signalling in mammalian cells . The results indicated that elevation of cGMP levels was stimulatory whereas inhibition of a putative Na+/H+ antiporter or calcium mobilization was inhibitory, and it is argued that high intracellular levels of cAMP may be inhibitory . Whilst the precise effects of the agents used on nematode cells remain to be established, these results provide a framework for the examination of the processes involved in the modification of the nematode surface which takes place immediately after the infection event.

Harefuah, 1993 Nov 15, 125(10), 347 - 9, 391
{Acanthamoebic keratitis}; Avni I et al.; Acanthamoebic keratitis is still a rare infection . It occurs in contact lens-wearers, especially when saline is prepared at home from contaminated tap water . There are periods of remission, and occasionally misleading findings resembling those of herpetic keratitis, which make the diagnosis difficult . The isolation of the acanthamoeba is not easy and special culture media are required . Early recognition and aggressive therapy with antiamebic medication and epithelial debridement, often in conjunction with penetrating keratoplasty, are needed . We describe the clinical course, laboratory diagnosis and treatment of 3 patients with acanthamoebic keratitis, 2 men aged 20 and 25, respectively and a women aged 42.

Arch Biochem Biophys, 1993 Nov 15, 307(1), 110 - 8
Fibronectin fragments alter matrix protein synthesis in cartilage tissue cultured in vitro; Xie D et al.; We reported earlier that fibronectin fragments (Fn-f) added to bovine articular cartilage cultured in serum-free culture causes marked protease expression with resultant proteoglycan (PG) degradation and release into the culture media . We have further characterized the effects of Fn-f by studies of the effects on proteoglycan, collagen, general protein, and DNA synthesis and reversibility of the cartilage damage . We report here that the most active Fn-f, a 29-kDa amino-terminal Fn-f, when added to a 1 microM concentration, depressed PG and general protein synthesis in cartilage by over 50% within 24 h, as measured by sulfate and methionine/cysteine incorporation, respectively . This same Fn-f decreased PG synthesis throughout the full thickness cartilage section as shown by autoradiography . PG and general protein synthesis were significantly depressed within 24 h by 29-kDa Fn-f concentrations as low as 10 nM . Synthesis rates were effected by 100-fold lower Fn-f concentrations than was induction of proteinases . Removal of the 29-kDa Fn-f allowed a gain to supernormal levels of PG and protein synthesis . Cartilage damaged to the extent of removal of over 50% of the total PG did not replace PG after over 4 weeks in 10% serum-Dulbecco's modified Eagle minimum with or without added TGF-b1 and rIGF-a . These data show that while the effects of Fn-f on elevating protease expression and depressing PG synthesis are reversible, the resultant cartilage damage is apparently irreversible in vitro . Therefore, if Fn-f-mediated cartilage damage occurs as part of cartilage disease processes, the pathologic effects would be quite significant.

J Immunol Methods, 1993 Nov 5, 166(1), 75 - 84
Analysis of active antibody concentration . Separation of affinity and concentration parameters; Karlsson R et al.; Antibody binding to surfaces with differing amounts of immobilised antigen was measured in a biosensor system using surface plasmon resonance detection . Binding rates obtained during the initial binding phase on high density antigen surfaces were proportional to antibody concentration and independent of antigen-antibody affinity . One antibody calibration curve covering the range from 0.5 to 160 nM (0.08-25 micrograms/ml) antibody was valid for IgG antibodies with different antigen specificities . To illustrate the use of this methodology active antibody concentrations were analysed in culture media and in rabbit serum.

Endocrinology, 1993 Nov, 133(5), 1990 - 8
Retinoic acid differentially regulates expression of surfactant-associated proteins in human fetal lung; Metzler MD et al.; Retinoic acid is known to play an essential role in maintaining the differentiation of a wide variety of epithelial cell types . However, its effects on the differentiation of lung alveolar epithelium have not been described . In the present study, we examined the effects of retinoic acid on the differentiation of human fetal lung tissue maintained in vitro . Human fetal lung explants were cultured in serum-free medium for 6 days in the absence or presence of all-trans retinoic acid at concentrations from 0.3 nM to 3 microM . Explant content of the surfactant-associated protein SP-A was measured using a specific enzyme-linked immunosorbent assay . Retinoic acid reduced SP-A protein levels in a concentration-dependent manner {analysis of variance (ANOVA), P < 0.01} . To evaluate possible cytotoxic effects of retinoic acid, culture media were assayed for lactate dehydrogenase (LDH), a cytoplasmic enzyme . LDH levels in media from retinoic acid-treated explants were not significantly different than LDH levels in media from control explants, indicating that retinoic acid is not cytotoxic in human fetal lung explants . Changes in messenger RNA (mRNA) levels for surfactant-associated proteins SP-A, SP-B, and SP-C were measured by Northern blot analysis . Retinoic acid reduced SP-A mRNA levels in a concentration-dependent manner (ANOVA, P < 0.02) and reduced SP-C mRNA levels at 3 microM . In contrast, retinoic acid increased SP-B mRNA levels in a concentration-dependent manner (ANOVA, P < 0.03) . Morphometric analysis showed that retinoic acid decreased epithelial volume density in the explants by approximately 17% and increased connective tissue volume density by approximately 20% when compared to dimethyl sulfoxide vehicle controls . These data indicate that retinoic acid regulates type II cell surfactant protein gene expression in human fetal lung tissue.

Prostaglandins Leukot Essent Fatty Acids, 1993 Nov, 49(5), 847 - 50
Prostaglandin E2 antagonizes gingival fibroblast proliferation stimulated by interleukin-1 beta; ElAttar TM et al.; Treatment of human gingival fibroblasts with prostaglandin E2 (PGE2) resulted in significant concentration-dependent inhibition in deoxyribonucleic acid (DNA) synthesis (8.40-37.89%), while indomethacin (INDO) (PG inhibitor), interleukin-1 beta (IL-1 beta) or IL-1 beta+INDO caused a significant and dose-dependent increase in DNA synthesis . Addition of PGE2 to culture media containing IL-1 beta and INDO caused a significant concentration-dependent reduction in IL-1 beta- and INDO-induced stimulation of DNA synthesis . The findings suggest that IL-1 beta and PGE2, which are also produced by fibroblasts, could play an important role in regulation of gingival tissue development and wound healing, and their modulation may have therapeutic potential.

Anticancer Res, 1993 Nov-Dec, 13(6A), 2355 - 60
In vitro estradiol-sensitivity characterization of the MCF-7, ZR-75, MDA-MB-231 and T47-D human breast neoplastic cell lines; Camby I et al.; Even if it seems that everything has been said about the influence of estradiol on cell proliferation in human breast cancer cell lines such as MCF-7, T47-D, ZR-75 and MDA-MB-231, in this study of the possible autocrine and/or paracrine role of 17 beta estradiol (E2) on the proliferation of human breast cancer cell lines we nevertheless offer some complementary information in this field of research . We exogenously stimulated the cell lines by the addition to the culture media of E2 and the anti-E2 antibody . The latter neutralizes the effects of any endogenous E2 . The cell proliferation was assessed by means of the MTT colorimetric test . We thus showed that the level of sensitivity of various breast cancer cell lines to estradiol in terms of cell proliferation depends on the experimental schedule chosen . Indeed, the addition of E2 to the culture media stimulated the growth of the the ZR-75 and T47-D cell lines conventionally described as estrogen-receptor positive (ER+) . For the MCF-7 cell line and the conventionally described as estrogen-receptor negative (ER-) MDA-MB-231 cell line, this is not the case . In sharp contrast, the addition to the culture media of the antibody neutralizing the biophysical activity of E2 sharply decreased the proliferation rate of the four cell lines under study . So these four cell lines in fact seem to be estradiol-sensitive . Thus, those which do not react to the addition of estradiol might use E2 in an autocrine and/or paracrine manner.

Atherosclerosis, 1993 Nov, 103(2), 279 - 90
Proteoglycans and endothelial barrier function: effect of linoleic acid exposure to porcine pulmonary artery endothelial cells; Ramasamy S et al.; Certain fatty acids induce changes in endothelial barrier function which may be mediated by alterations in normal proteoglycan synthesis/metabolism . To test this hypothesis, pulmonary artery derived endothelial cells were treated with media supplemented with linoleic acid (18:2), and/or a known proteoglycan synthesis inhibitor, beta-D-xyloside . Independent exposure to 1 mM beta-D-xyloside or 90 microM 18:2 increased albumin transfer, i.e., decreased barrier function, when compared with control cultures . 18:2 and beta-D-xyloside increased albumin transfer additively, suggesting that the mechanisms by which 18:2 and beta-D-xyloside alter the proteoglycan metabolism are different . Compared with the control group, treatment with 18:2 inhibited proteoglycan synthesis, decreased anionic properties of heparan sulfate proteoglycans in the cell monolayers and caused the release of a unique chondroitin sulfate proteoglycan into the culture media . Treatment with beta-D-xyloside caused an increased incorporation of radioactive sulfate into glycosaminoglycans but inhibited proteoglycan synthesis . These results suggest that the fatty acid- and beta-D-xyloside-induced impairment in endothelial barrier function may involve changes in the synthesis, release and physicochemical properties of proteoglycans.

J Bone Miner Res, 1993 Nov, 8(11), 1301 - 9
Stimulatory effect of TGF-beta on anionic glycoconjugate synthesis by rat calvarial cells: specificity, uncoupling of cell density dependence, and modulation by chondroitin sulfate; Anastassiades TP et al.; Anchorage-dependent cultures of a population of cells derived from the outer part of the rat calvaria demonstrated decreased net accumulation of radiolabeled chondroitin sulfate (CS) and hyaluronic acid (HA) per cell as the cell density of the cultures increased . The addition of TGF-beta 1 resulted in large stimulations of the net CS, but not of the net HA, accumulating in the medium at all cell densities and an abolition of the density-dependent effect . These effects were largely due to increases in newly synthesized CS appearing in the medium . Supplementation of the culture media with CS had complex but relatively small effects on the stimulation of the net accumulation of radiolabeled medium CS by TGF-beta 1 . The addition of TGF-beta 1 also resulted in a biphasic effect on cell growth that depended on the plating density, but cell growth differences could not account for the marked stimulation of CS synthesis by TGF-beta 1 . Experiments with cycloheximide and beta-xyloside and isolation of the intact anionic glycoconjugates (AG) indicated that although synthesis of core protein was the limiting factor in CS synthesis, TGF-beta 1 stimulated the synthesis of CS chain when sufficient beta-xyloside acceptor was available . The overall results suggest that, in this cell system, the action of TGF-beta 1 on the synthesis of the major extracellular AGs is characterized by a relatively specific upregulation of CS proteoglycan (PG) synthesis and an uncoupling of the inhibitory effect of high cell density on CS PG synthesis.

J Clin Microbiol, 1993 Nov, 31(11), 3050 - 2
Growth in serum-free medium improves isolation of Chlamydia pneumoniae; Maass M et al.; Infectivity titers were determined for eight Chlamydia pneumoniae strains simultaneously grown in serum-free and serum-supplemented cell culture media . Use of serum-free medium resulted in a 10- to 50-fold increase in the susceptibility of HL cells to chlamydial infection . Comparative primary isolation of a wild-type strain also produced higher inclusion counts in a serum-free environment . Serum-free cultivation is recommended to increase the efficiency of C . pneumoniae isolation from clinical material and to permit elementary body purification without interference caused by serum components.

Magn Reson Med, 1993 Nov, 30(5), 617 - 25
Intracellular labeling of T-cells with superparamagnetic contrast agents; Yeh TC et al.; Isolated rat T-cells have been labeled intracellularly, using endocytosis uptake of two superparamagnetic contrast agents, AquaMag100 and BMS180549, which are both iron-oxide particles coated with dextran . No deterioration of cell proliferation response to mitogen stimulation was observed after labeling with either superparamagnetic contrast agent . AquaMag100 particles show aggregation and co-precipitation in culture media for T-cells . BMS180549 particles not only produce no observable aggregation or co-precipitation, but also have a higher efficiency for labeling T-cells than AquaMag100 . The efficiency of cell labeling was determined by measuring the decrease in the spin-spin relaxation time of the water proton in cell samples containing 1 x 10(7) labeled T-cells/milliliter of 2% w/w gelatin . After optimization of the labeling procedures, a shortening of the spin-spin relaxation time by a factor of approximately 7 to 10 has been demonstrated . Under the present experimental conditions, the up-regulation of low density lipoprotein receptor does not increase the labeling efficiency by endocytosis . Our results suggest that intracellular labeling of specific cell types can be achieved with good efficiency and the labeled cells can be detected by magnetic resonance imaging in rat testicles in vivo.

Pancreas, 1993 Nov, 8(6), 719 - 25
Chronic hyperglycemia and the human fetal beta cell; Tuch BE et al.; It is well known that the ability of the immature rodent fetal beta cell to release insulin in response to a glucose challenge can be enhanced by chronic exposure to a high concentration of glucose in vitro . It might be thought that the human fetal beta cell would mature similarly in vitro, because neonates born of diabetic mothers release insulin in a more mature manner than normal infants . Using an organ culture of human fetal pancreatic explants, we have examined this possibility by exposing the tissue to 0-30 mM glucose . Six weeks of exposure of pancreatic explants to as high a concentration of glucose as 30 mM did not cause significant enhancement of the insulinogenic response to an acute challenge with 20 mM glucose . In contrast, chronic insulin release was enhanced, although culture medium containing 2.8 mM glucose was equally as efficacious as that with 30 mM glucose . Just as with insulin, proinsulin levels in the culture media containing no glucose also were suppressed . Degranulation of the beta cell exposed to high concentrations of glucose did not occur, the insulin content of the explants at the end of culture being enhanced in those maintained in 5.6-30 mM but not 2.8 mM glucose . Desensitization to the acute stimulatory effect of 10 mM theophylline did not eventuate, even in explants exposed to 30 mM glucose . In contrast to the human explants, rat fetal pancreatic explants did mature when exposed to 11.2 mM glucose for 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

Fertil Steril, 1993 Nov, 60(5), 876 - 80
In vitro deleterious effect of hypoxanthine in Ham's Nutrient Mixture F-10 culture medium on human oocyte fertilization and early embryonic development; Bastias MC et al.; OBJECTIVE: To determine whether hypoxanthine in Ham's Nutrient Mixture F-10 (GIBCO, Grand Island, NY) culture medium affects murine embryo development or the outcome of patients undergoing IVF-ET . DESIGN: For the two-cell embryo bioassay, embryos from each female were equally distributed and incubated in Ham's F-10 with or without hypoxanthine supplementation for up to 72 hours . To assess the effect of hypoxanthine in human IVF-ET, oocytes, sperm, and embryos were cultured in Ham's F-10 medium with or without hypoxanthine . Fertilization and embryo cleavage were assessed at 18 and 40 hours, respectively, after insemination . SETTING: University medical research laboratory . PATIENTS, PARTICIPANTS: Nine couples undergoing IVF-ET . RESULTS: Two-cell mouse embryos incubated for 24 hours without hypoxanthine developed 40% to morula, compared with 6.5% for the hypoxanthine group . At 72 hours, 99.5% of the embryos without hypoxanthine reached the expanded blastocyst stage with 65% of them exhibiting hatching, compared with 72% and 19.5%, respectively, for the group with hypoxanthine . Human oocytes cultured in Ham's F-10 without hypoxanthine showed higher fertilization rates than the group incubated in the presence of hypoxanthine (69% versus 53%) . The proportion of cleaved embryos was not different between the two culture media; however, the rate of embryos cleaving without cytoplasmic fragments was significantly higher in the group without hypoxanthine (75% versus 35%) . CONCLUSION: Ham's F-10 medium containing hypoxanthine significantly reduced embryo development in the two-cell mouse embryo bioassay . Hypoxanthine in culture medium for IVF-ET may have a deleterious effect on human gametes, leading to decreased fertilization and increased incidence of cytoplasmic fragments in the conceptuses.

Stem Cells, 1993 Nov, 11(6), 519 - 27
Multi-center collaborative in vitro studies using a human cancer cell line: the EORTC Preclinical Therapeutic Models Group experience; Eliason J; The EORTC Preclinical Therapeutic Models Group (PTMG), formerly known as the Clonogenic Assay Screening Study Group (CASSG), began experiments in 1982 to investigate the feasibility of doing collaborative studies using in vitro clonogenic assays . The human colon adenocarcinoma cell line WiDr was selected as the model with which all participating laboratories would work . During the course of these studies, representatives from 34 different institutions located in 10 European countries participated . The first two collaborative experiments attempted to standardize the assay techniques using a double layer agar clonogenic assay . The results indicated that it was not possible to truly standardize these techniques in an international setting . The overall results demonstrated that the WiDr cell line grew well under a variety of conditions and that cloning efficiencies were independent of cell concentration, provided that a concentration of about 3,000 cells/ml was not exceeded . For the next series of protocols, the overall objectives were modified so that each laboratory could use its preferred assay technique whereas the WiDr cells were standardized by being expanded in a single center and sent to participants as viable cells . Analysis of the pooled results indicated that there was steady improvement in reproducibility for the group as a whole with repetition of the protocol . Drug dose responses with doxorubicin and etoposide showed good reproducibility from experiment to experiment . The results with cisplatin suggested that the sensitivity of this cell line may be affected by the presence of reduced sulfhydryl in some tissue culture media . The overall experience of the group indicates that use of a cancer cell line can provide the framework within which collaborative in vitro studies can be performed, provided that the initial conditions such as cell and drug concentrations are carefully determined in advance through preliminary studies.

J Reprod Fertil, 1993 Nov, 99(2), 467 - 70
Influence of the anti-androgen hydroxyflutamide on in vitro development of mouse embryos; Yallampalli C et al.; Hydroxyflutamide is a potent nonsteroidal anti-androgenic drug extensively used in laboratory investigations . Our present studies were aimed at determining whether this anti-androgen modulates development of embryos in vitro . One-cell and two-cell mouse embryos were collected by flushing the oviducts and cultured in defined culture media in the presence or absence of various doses of hydroxyflutamide . The development of embryos from one-cell and two-cell to blastocyst stage was assessed . Hydroxyflutamide caused a dose-dependent (0-100 micrograms ml-1) inhibition of development of both one-cell and two-cell embryos; 100% inhibition was observed at 20 micrograms ml-1 . The adverse effects of hydroxyflutamide on two-cell embryo development were completely reversed by testosterone in a dose-dependent manner, but not by oestradiol and progesterone . These results indicate that the antiandrogen hydroxyflutamide inhibits early embryo development suggesting that it may be useful during the preimplantation period for preventing conception.

Fertil Steril, 1993 Nov, 60(5), 839 - 51
Polypeptides synthesized and released by human endometriosis differ from those of the uterine endometrium in cell and tissue explant culture; Sharpe KL et al.; OBJECTIVE: To identify and compare the pattern of polypeptide synthesis and release of endometriosis with that of the normal endometrium in culture . DESIGN: Endometriosis and endometrial biopsy specimens were obtained from women presenting for a variety of diagnostic and therapeutic examinations . Specimens were cultured as isolated epithelial and stromal cells and tissue explants with L-{35S}methionine . De novo synthesized proteins were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, and Western blot analysis . RESULTS: Five major deviations in protein synthesis and secretion were noted when comparing endometriotic and endometrial culture media . Endometriosis synthesized and secreted two proteins of stromal cell origin that were not produced by normal endometrium: endometriosis protein group I (M(r) 40,000 to 55,000; pI 4.0 to 5.2) and endometriosis protein group II (M(r) 30,000 to 32,000; pI 7.0 to 9.0) . Conversely, endometriosis lacked the ability to secrete or asynchronously secreted three groups of secretory phase endometrial proteins in culture: endometrial protein group I (M(r) 25,000 to 27,000; pI 4.5 to 5.5) and endometrial protein group II (M(r) 68,000 to 72,000; pI 3.0 to 3.2) were endometrial epithelial cell products whereas endometrial protein group III (M(r) 70,000; pI 5.7) was of endometrial stromal cell origin . CONCLUSIONS: Unique characteristics in endometriosis protein synthesis and secretion as compared with the endometrium indicate biochemical dissimilarities between these tissues, which may be used to develop diagnostic markers and gain insight into the etiology and/or pathophysiology of the disease.

Parasite Immunol, 1993 Nov, 15(11), 613 - 8
Mice immunized with a synthetic peptide construct corresponding to an epitope present on a Plasmodium falciparum antigen are protected against Plasmodium chabaudi challenge; Lord R et al.; Inhibitory monoclonal antibody (MoAb) 8E7/55 recognizes a parasitophorous vacuole membrane (PVM) antigen in Plasmodium falciparum . Previous studies have identified the epitope, DNNLVSGP, recognized by the MoAb . A synthetic peptide containing this sequence was synthesized and coupled to diphtheria toxoid (DT) and was found capable of generating antibodies when used as an immunogen in mice which recognize the native antigen exp-1 . In this study we demonstrate the ability of the MoAb and antisera generated against the peptide construct to recognize a 54 kD PVM antigen in Plasmodium chabaudi . The P . chabaudi antigen is synthesized in trophozoites and released to the surrounding culture media outside the parasitized erythrocyte . Mice immunized with the peptide conjugate are protected when challenged with a lethal strain of P . chabaudi . Protection in the mice correlated with the antibody titre prior to challenge . If the PVM antigen from P . chabaudi is a homologue of exp-1 from P . falciparum, then these experiments may provide a guide to the antibody titres required in human trials before antibody mediated protection could be expected . The discovery that a PVM localized antigen is secreted into the surrounding in vitro culture media provides us with a valuable model system for further investigation of protein trafficking pathways in malaria-infected erythrocytes.

Jpn J Pharmacol, 1993 Nov, 63(3), 361 - 7
Dexamethasone inhibits nitric oxide synthase mRNA induction by interleukin-1 alpha and tumor necrosis factor-alpha in vascular smooth muscle cells; Marumo T et al.; The effects of interleukin-1 alpha, tumor necrosis factor-alpha and dexamethasone on the induction of nitric oxide synthase mRNA in rat aortic smooth muscle cells were studied . Neither interleukin-1 alpha (up to 100 U/ml) nor tumor necrosis factor-alpha (up to 5000 U/ml) was capable of inducing nitrite/nitrate production and nitric oxide synthase mRNA in smooth muscle cells . In contrast, treatment for 12 hr or longer with a combination of the two synergistically induced nitrite/nitrate and cyclic GMP production in cell culture media and nitric oxide synthase mRNA, both of which were prevented by dexamethasone . Contamination with bacterial lipopolysaccharide, which may affect the induction of nitric oxide synthase, was below 30 pg/ml in all experiments . Our findings show that dexamethasone and these cytokines regulate the induction of nitric oxide synthase at the mRNA level in vascular smooth muscle cells.

Gen Comp Endocrinol, 1993 Nov, 92(2), 179 - 88
Growth hormone receptors in avian epiphyseal growth-plate chondrocytes; Monsonego E et al.; Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plate cartilage by Northern blot and hybridization using the avian GH-R probe . A single transcript of approximately 5.2 kb was demonstrated in cultured growth-plate chondrocytes as well as in growth-plate extracts . GH receptor gene expression was inhibited by chicken GH (cGH) in a dose- and time-dependent manner . Chicken GH was more potent in down-regulating the GH-R gene expression than hGH, but on the other hand cGH exhibited a lower affinity to avian chondrocytes receptor than did the human hormone . Addition of ascorbic acid to the culture media caused cell differentiation: induction of alkaline phosphatase activity and attenuation of collagen type II gene expression . No differences in the GH-R gene expression were observed in the nondifferentiated cells compared with the differentiated cells . Chicken GH did not form any complex with the purified hGH binding protein (hGHBP), did not bind to human lymphocytes GH receptor, and did not affect Nb2 cell proliferation . These systems represent somatogenic and lactogenic types of GH receptors, respectively . In summary, avian growth-plate chondrocytes in situ and in culture exhibit GH-R and these receptors are capable of binding GH . Thus, the failure of GH to affect avian chondrocytes' proliferation was not due to either the absence of receptors on the cell membrane or to a lack in its binding activity, but rather may be due to events farther downstream.

FEBS Lett, 1993 Oct 11, 332(1-2), 99 - 104
Prevention of the induced synthesis and secretion of group II phospholipase A2 by brefeldin A; Sanchez RM et al.; Brefeldin A (BFA) has previously been shown to block protein secretion and to cause dismantling of the Golgi cisternae in many cultured cell lines . BFA was found to prevent the induced synthesis and secretion of 14 kDa group II phospholipase A2 (PLA2) in rat mesangial cells . Furthermore, BFA inhibited total protein synthesis although PLA2 appeared to be more sensitive to the effect of this compound than total protein synthesis assessed by amino acid incorporation . BFA was unable to block protein synthesis or PLA2 activity in the cell completely but secretion of enzymatic activity and PLA2 protein into the cell culture media was totally inhibited.

J Surg Res, 1993 Oct, 55(4), 411 - 5
Effect of hyaluronic acid on rabbit profundus flexor tendon healing in vitro; Salti NI et al.; We performed an in-depth biomechanical evaluation of the effect of hyaluronic acid (HA) on the healing of rabbit profundus tendons cultured in vitro . Seventy-eight flexor tendons from 13 rabbits were transected and reapproximated at their Zone II midpoints . Tendons were divided into left and right forepaw groups . Each tendon from the left forepaw group was incubated in one of four possible culture media: control (no HA), low (0.1 mg/ml), medium (0.5 mg/ml), or high (1.0 mg/ml) HA media . HA was added on the first day of incubation . Each tendon from the right forepaw group was cultured in low, medium, or high concentrations of HA, but HA was added after 1 week of incubation in control media . All tendons were cultured for 8 weeks, after which time tenorrhaphies were disrupted and the following biomechanical parameters were determined: apparent maximum stress, apparent strain at apparent maximum stress, normalized energy absorption, and tangent modulus before failure . Comparisons using these parameters showed no statistically significant differences among the various tendon groups . We believe this is the first study of its kind to show no effect of hyaluronic acid on the functional strength of tendon after healing in vitro.

J Surg Res, 1993 Oct, 55(4), 397 - 403
Modulation of cytotoxic activity of resident macrophages by postsurgical macrophages; Kuraoka S et al.; The purpose of this study was to determine if the secretion of cytotoxic molecules, such as tumor necrosis factor or toxic oxygen molecules, by resident peritoneal macrophages is modulated by postsurgical macrophages elicited by peritoneal trauma . Resident macrophages were collected from nonsurgical rabbits and cultured in vitro with either spent media from cultures of postsurgical macrophages harvested at various times or with varying concentrations of standard cytokines . Superoxide anion (O2-) production of resident macrophages increased with exposure to spent culture media from macrophages obtained after intestinal reanastomosis (3, 6, 12, 24 hr) . This increase reached maximal levels by 6 hr after surgery and thereafter decreased to resident levels by 24 hr after surgery . Exposure of resident macrophages to spent media from cells collected after peritoneal sidewall abrasion (1, 3, 5, 7, 10, 14 days) elevated the production of O2- on Postsurgical Days 3 and 5; however, no effect was observed following exposure to spent media of macrophages harvested on Postsurgical Day 14 . Interleukin-1 alpha (IL-1 alpha), transforming growth factor beta (TGF-beta), and tumor necrosis factor alpha (TNF alpha) stimulated phorbol ester-induced O2- production by resident macrophages in a concentration-dependent manner . The secretion of TNF activity by resident macrophages increased following exposure to spent media of macrophages harvested 6 to 24 hr after intestinal surgery . IL-1 alpha, TGF-beta, and TNF alpha elevated the secretion of TNF activity by resident macrophages in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurochem, 1993 Oct, 61(4), 1323 - 32
In vivo production and release of acetylcholine from primary fibroblasts genetically modified to express choline acetyltransferase; Fisher LJ et al.; Primary rat fibroblasts genetically modified to express Drosophila choline acetyltransferase (dChAT) synthesize and release acetylcholine (ACh) in vitro . The ACh produced from the transduced fibroblasts was found to be enhanced by increasing amounts of choline chloride in the culture media . These dChAT-expressing cells were then implanted into the intact hippocampus of adult rats and in vivo microdialysis was performed 7-10 days after grafting to assess the ability of the cells to produce ACh and respond to exogenous choline in vivo . Samples collected from anesthetized rats revealed fourfold higher levels of ACh around dChAT grafts than from either non-grafted or control-grafted hippocampi . Localized choline infusion (200 microM) through the dialysis probes was found to induce a selective twofold increase in ACh release only from the dChAT-expressing fibroblasts . These results indicate not only that dChAT-expressing fibroblasts continue to synthesize and secrete ACh for at least 10 days after intracerebral grafting, but that the levels of ACh can be manipulated in vivo . The ability to regulate products within genetically modified cells in vivo may provide a powerful avenue for exploring the role of discrete substances within the CNS.

Teratology, 1993 Oct, 48(4), 285 - 97
Toxicokinetics and structure-activity relationships of nine para-substituted phenols in rat embryos in vitro; Fisher HL et al.; The purpose of this study was to examine the toxicokinetics of embryo uptake following exposure to a variety of chemically related phenols in rat embryo culture . The uptake of nine radiolabeled para-substituted phenols by day 10 (9-13 somite stage) rat embryos in vitro was determined from 1 to 42 hrs after being placed in culture media containing various phenols . Uptake was rapid, having a half-life of 3 hr or less, with 7 of the nine compounds having uptake half-times of less than one hour . The equilibrium concentration in the embryo ranged from 53 to 136% of the media concentration, indicating only a factor of 2 in maximum discrimination against the compound for any of the phenols studied . The fraction of radioactivity remaining unbound in the media decreased with increasing log P (octanol/water partition coefficient) . The binding was calculated to be 50% for log P = 1.77 from the fitted regression equation and decreased by a factor of 5.9 for every decade increase in P . When hepatocytes were also present in the media the equilibrium concentration in the embryos was less than when hepatocytes were absent . With the limited data, four of the phenols appeared to have no (i.e., zero) equilibrium level when hepatocytes were present . Thus the metabolites produced by the hepatocytes appeared to have less affinity for the embryo than the parent phenol . Toxicodynamic information as given by the effective concentration of the phenol in the embryo to cause somite or tail teratological effects was best predicted by the measured unbound fraction.

Pediatr Nephrol, 1993 Oct, 7(5), 616 - 20
Growth factors and kidney development; Hammerman MR et al.; The formation of the metanephric kidney is dependent upon the timed and sequential expression of a number of polypeptide growth factors . To shed light on the participation of members of the insulin-like growth factor (IGF) and epidermal growth factor/transforming growth factor-alpha (EGF/TF-alpha) families, we measured the synthesis of IGF-I, IGF-II, EGF and TGF-alpha by developing rat metanephroi in organ culture and determined the effect of anti-growth factor antibodies on growth and development . IGF-I, IGF-II and TGF-alpha were produced by metanephroi and released into culture media . We could detect no EGF . Inclusion of anti-IGF-I, anti-IGF-II, anti-IGF-II receptor or anti-TGF-alpha antibodies in organ cultures inhibited growth and development of metanephroi . Our findings suggest that both members of the IGF family and TGF-alpha are produced within the developing metanephros and promote renal organogenesis.

J Geriatr Psychiatry Neurol, 1993 Oct-Dec, 6(4), 239 - 44
Aluminum induces neurite elongation and sprouting in cultured hippocampal neurons; Uemura E et al.; It has been reported that the aluminum content in the human brain increases with age, and it is particularly high in those with Alzheimer's disease . In this study, we found that a low aluminum concentration (100 mumol/L) in the culture media of opossum hippocampal neurons can induce extensive neurite outgrowth (ie, elongation and branching of neurites) and sprouting (ie, outgrowth of filiform processes from neurite varicosities) within 48 hours . Such changes in neurite morphology were remarkably similar to those described in the aged or Alzheimer's disease brain . Neurites that responded to aluminum varied greatly in length, thickness, and branching pattern . Many neurites appeared to have no clear directional growth pattern because they frequently changed their course and formed a meshwork of neurites with others originating from the same cell body . Sprouting neurites varied in length, thickness, and branching pattern, but they always originated from a globular enlargement of neurites along the neurite shaft or at the terminal end . Such growth pattern and extensive sprouting of neurites did not fit the growth pattern displayed by the control neurons . Our findings suggest that aluminum may be involved in the neuronal remodeling characteristic of aging and Alzheimer's disease.

Anal Biochem, 1993 Oct, 214(1), 100 - 5
Determination of integrins on cells by cell adhesion to antibodies; Holmes E et al.; An assay for the determination of relative concentrations of integrins on clonal cultured cells is described . Unpurified monoclonal antibodies to integrin subunits, present in ascites or hybridoma culture media, are immobilized on a plastic surface via goat antibodies to mouse IgG . Cell attachment to the integrin-coated substrate is quantitated after fixation and staining of the cells . The assay is specific and very sensitive; only cells with the relevant integrins attach to antibody-coated substrates and concentrations of antibody as low as 1-10 ng/ml are sufficient . Titrations of the antibodies on the solid phase allow the estimation of the relative amounts of integrins on different cell types . The results with this assay correlate well with results obtained by flow cytometry.

Curr Opin Obstet Gynecol, 1993 Oct, 5(5), 615 - 22
GIFT, ZIFT, and related techniques; Abyholm T et al.; This review focuses on the theoretical backgrounds for tubal gamete and zygote/embryo transfer, as well as the clinical results of gamete intrafallopian transfer (GIFT), which are compared with other non-fertilization procedures in infertile women with patent fallopian tubes . While GIFT and zygote intrafallopian transfer (ZIFT) probably result in a more synchronized entry of embryos into the uterine cavity, prospective, randomized studies have not shown these methods to be preferable to conventional in-vitro fertilization and embryo transfer . Nevertheless, co-culture with various cell types seems to yield more viable embryos with a high rate of implantation . The promising results with co-culture do not seem to be a cell- or species-specific phenomenon . This non-specific positive or negative conditioning effect of co-culture on embryo quality indicates that more optimal culture media for in-vitro fertilization can probably be devised . The requirements of laparoscopy and general anesthesia with GIFT have prompted the development of simpler methods based on fertilization in vivo . Various methods of artificial insemination combined with controlled ovarian hyperstimulation yield comparable results with GIFT in unexplained infertility . In endometriosis, GIFT seems to give better results compared with insemination techniques . Less invasive transcervical gamete and embryo transfer techniques have now been established, obviating the need for operating theater facilities.

J Physiol, 1993 Oct, 470, 501 - 20
Mitogenic factors regulate ion channels in Schwann cells cultured from newborn rat sciatic nerve; Wilson GF et al.; 1 . Patch clamp studies were carried out in Schwann cells cultured from newborn rat sciatic nerve to determine the effects of mitogens on voltage-gated currents without the confounding influences of axonal contact and myelin present in vivo . The relevance of the various Schwann cell currents to proliferation was assessed using assays of {3H}thymidine incorporation . 2 . Treatment of cultured Schwann cells with known mitogens, namely axon fragments (AF), myelin fragments (MF), or glial growth factor in combination with forskolin (GGF+F), increased the magnitudes of delayed rectifying potassium (K+) and sodium (Na+) currents . 3 . In both control and mitogen-treated cells, the magnitude of net outward current paralleled clearly the magnitude of the cells' proliferative response . 4 . The K+ channel-blocking quaternary ammonium ions, tetrabutylammonium (TBuA), tetrapentylammonium (TPeA) and tetrahexylammonium (THeA), but not the Na+ channel blocker tetrodotoxin (TTX), reduced proliferation in a dose-dependent fashion offering further evidence for a role for K+ channels in Schwann cell proliferation . 5 . Voltage-gated chloride (Cl-) currents were observed in both control and mitogen-treated cells . Addition of the Cl- channel blockers, 4-acetamido-4'-isocyanatostilbene-2,2'-disulphonate (SITS) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS), to the culture media enhanced proliferation . 6 . The possible intermediary role of the Schwann cell resting potential was explored in ion substitution experiments by increasing the K+ concentration of the media and by adding ouabain . Both manipulations inhibited Schwann cell mitosis . 7 . Comparison of the expression of functional ion channels in vitro with that previously described for Schwann cells in vivo suggests a difference in the Schwann cell response to the membrane fragment mitogens and their intact counterparts in regard to the regulation of ion channels . MF up-regulates the number of functional channels, whereas the elaboration of myelin (or a factor related to its presence) in vivo appears to down-regulate channel expression, at the cell soma of myelinating Schwann cells . In addition, axonal contact may be required for normal expression of functional inwardly rectifying K+ channels.

J Pharmacol Toxicol Methods, 1993 Oct, 30(2), 111 - 5
An in vitro pharmacological model of vascular smooth muscle; Hussain T et al.; In order to develop an in vitro model of vascular tissue for pharmacological studies, segments of porcine coronary arteries were incubated in culture media under sterile conditions in cell culture incubator at 37 degrees C with 95% O2 + 5% CO2 . After 3 days of incubation, changes in isometric tension were measured in vascular rings and compared with the fresh tissue . KCl (10-75 mM) and prostaglandin F2 alpha (1-20 microM) produced a similar concentration-dependent contraction in the incubated and fresh arteries . The concentration-dependent relaxation curves produced by 2-chloroadenosine (10(-8) to 10(-4) M) and isoproterenol (10(-8) to 10(-5) M) were unaltered in the incubated tissue versus fresh . Similarly, the relaxation responses to forskolin and sodium nitroprusside (10(-8) to 10(-5) M) were unaffected in the incubated arteries . The relaxations produced by substance P (10(-12) to 10(-8) M) and bradykinin (10(-7) M)--the endothelium-dependent agents--were also unaltered in the incubated rings versus fresh . Therefore, we conclude that after the incubation of porcine coronary artery for 3 days, the contraction/relaxation responses to various agonists acting through different mechanisms were unaltered in porcine coronary artery . This in vitro model of vascular smooth muscle provides a potential for pharmacological and toxicological studies.

Ann N Y Acad Sci, 1993 Sep 24, 695, 278 - 84
Cells engineered to produce acetylcholine: therapeutic potential for Alzheimer's disease; Fisher LJ et al.; Alzheimer's disease (AD) is a debilitating disorder of the central nervous system which may affect up to 50% of the population over the age of 85 years . The etiology of AD is unknown and there is currently no cure for the disease . Well-documented losses in cholinergic and other neurotransmitter systems have provided a focal point for attempting pharmacological interventions in AD to ameliorate some of the cognitive deficits that occur . However, current systemic strategies have met with limited success . An alternative strategy, that has been pursued in animal models of neurodegenerative disease, is to augment neurotransmitter function within the brain through tissue transplantation . Such implants have an advantage over conventional drug therapies in that the cells can be precisely placed within compromised areas of the brain . We have pursued a strategy of designing cells, through the use of molecular biology techniques, to produce neurotrophic factors and neurotransmitters . Recently, we developed a primary fibroblast cell line that was genetically modified to express choline acetyltransferase (ChAT) . In vitro, these cells produced and released acetylcholine at levels that varied with the amount of choline in the culture media . When implanted into the hippocampus of rats, the in vivo microdialysis technique revealed that the ChAT-expressing fibroblasts continued to produce and release acetylcholine after grafting . Most importantly, the levels of acetylcholine synthesized by the cells could be regulated by the localized infusion of choline in the vicinity of the grafts . These results confirmed previous work which indicated that engineered fibroblasts provide an effective delivery vehicle of different substances to the brain . While the intracerebral implantation of genetically modified cells will not cure AD, the continuing development of this strategy may ultimately provide a powerful approach for ameliorating the devastating cognitive impairments which are a hallmark of this disease.

Metabolism, 1993 Sep, 42(9), 1077 - 80
Dichloroacetic acid accelerates initial development of 2-cell murine embryos in vitro; Penzias AS et al.; Preimplantation embryos up to the 8-cell stage of development use lactate and pyruvate but not glucose or Krebs cycle intermediates to support growth, development, and cleavage . The dominant effect of dichloroacetic acid (DCA) is the irreversible stimulation of pyruvate dehydrogenase (PDH) activity, thus accelerating the oxidative metabolism of pyruvate and lactate . To test the hypothesis that early induction of oxidative metabolism in 2-cell murine embryos accelerates preimplantation embryo cleavage rates, female B6C3F1 mice at 6 to 8 weeks of age were superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and mated . All 2-cell stage embryos were randomly assigned to culture media with or without 130 micrograms/mL DCA . The developmental stage of all embryos was then noted every 24 hours for a total of 72 hours . Chi-square analysis and the method of average rank sum were used to compare the distribution of embryos at each observation point . At 24 hours, DCA-exposed embryos had achieved an advanced stage of growth and development relative to controls (average rank sum, P = .026; chi-square distribution, P = .047) . Subsequently, at 48 and 72 hours, neither the average rank sum nor the chi-square distribution was different . Our data suggest that DCA accelerates early growth and development of murine embryos before implantation, possibly through the early induction of oxidative metabolism.

Cell Biochem Funct, 1993 Sep, 11(3), 201 - 9
Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells; Chu AJ et al.; The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding {3H}choline, {14C}ethanolamine or {14C}glycerol to the culture media; incorporation into phospholipid (PL) increased with time . The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 microM 18:1 (n - 9), 18:2 (n - 6), 18:3 (n - 3), 20:4 (n - 6) and 20:5 (n - 3) . The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of {3H}thymidine and {3H}leucine . Total cellular uptake of radioactive precursors remained unaffected by all the treatments . Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of {3H}choline into PL, while no significant effect was detected with the other UFAs . 18:3, 20:4 and 20:5 depressed the incorporation of {14C}ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively . However, there was no redistribution of label with any of the treatments . 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis . In addition, the incorporation from {14C}glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5 . Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n - 3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.

Lab Invest, 1993 Sep, 69(3), 305 - 11
Expression of 72 kilodalton and 92 kilodalton type IV collagenase in malignant fibrous histiocytomas and dermatofibromas; Soini Y et al.; BACKGROUND: In previous studies, it has been shown that the stromal cells of epithelial tumors are capable of synthesizing 72 and 92 kilodalton (kd) type IV collagenase mRNA . The mRNA synthesis of these collagenases in mesenchymal tumors has not been extensively studied, however . This study was undertaken to explore the synthesis of 72 and 92 kd type IV collagenase mRNAs in malignant and benign fibrous histiocytomas and its correlation with the known biologic behavior of these tumors . EXPERIMENTAL DESIGN: The synthesis of 72 kd and 92 kd type IV collagenase mRNA was studied in 10 malignant fibrous histiocytomas (MFHs) and 7 dermatofibromas using in situ hybridization methods . The tumors were also studied with a commercial monoclonal antibody to the 72 kd type IV collagenase . Additionally, three dermatofibromas were studied with an antibody to the 92 kd type IV collagenase . The media of three cell lines (MFH, dermatofibroma and skin fibroblast cell line) were also analyzed by zymography assay . RESULTS: The results revealed mRNA for the 72 kd and 92 kd collagenases in tumor cells of both MFHs and dermatofibromas . Also intracytoplasmic immunoreactivity for the 72 kd type IV collagenase could be seen in all the tumors . In the zymography assay, 72 kd type IV collagenase activity was detected in the culture media of all the cell lines tested, but activity for the 92 kd enzyme was only seen in the MFH . However, immunoreactivity for the antibody to the 92 kd type IV collagenase was also seen in dermatofibromas . CONCLUSIONS: The findings indicate that MFHs and dermatofibromas produce type IV collagenases . The synthesis of the mRNAs for both 72 kd and 92 kd type IV collagenase was quantitatively similar in MFHs and dermatofibromas indicating that there is no correlation between the biologic behavior of the tumors and the synthesis of these substances . Therefore, additional factors other than synthesis of type IV collagenases by tumor cells, must be involved in the process of spread and invasion of tumor cells into the neighbouring tissues.

Am J Trop Med Hyg, 1993 Sep, 49(3), 335 - 40
Cultivation of Plasmodium falciparum parasites in a serum-free medium; Ofulla AV et al.; The elimination of serum from Plasmodium falciparum culture media could decrease costs, enhance procurement, and improve the feasibility of large-scale production of parasite material . We provide a semi-defined, serum-free formulation, of commercially available constituents that supports P . falciparum parasite growth at rates comparable with those obtained with serum-supplemented media . The medium is composed of RPMI 1640 to which HEPES, extra glucose, bicarbonate, and hypoxanthine have been added . Bovine albumin and serum-derived, lipids-cholesterol-rich mixture are then used in place of serum.

J Immunol, 1993 Sep 1, 151(5), 2613 - 22
The soluble pool of beta 2-microglobulin free HLA class I alpha-chains . Qualitative and quantitative characterization; Pickl WF et al.; Previous studies have demonstrated that HLA class I heterodimers are present in plasma and cell culture supernatants . They can be precipitated by mAb the binding of which is dependent on the proper association of the polymorphic alpha-chain with beta 2-microglobulin (beta 2-m) . The molecular mass of the alpha-chain ranges from 45 to 35 kDa with a number of intermediate products . We report on the identification of 35-kDa soluble beta 2-m free HLA class I H chains immunoprecipitated by mAb LA45 from cell culture media of activated B and T cells . Furthermore, a peptide-based competitive immunosorbent assay was established to determine the amounts of soluble HLA class I alpha-chains . By means of this assay, we formally proved the specificity of mAb LA45 for a linear epitope on HLA class I H chains centered on residues arginine-asparagine at positions 62 and 63 of the alpha 1-domain . PHA or rIL-2 were identified as efficient stimuli for PBMC leading to the generation of soluble beta 2-m free HLA class I H chains . Testing of cell lines representing distinct stages of hematopoietic differentiation demonstrated a significant correlation between cell surface expression of beta 2-m free HLA class I H chains and amounts of soluble LA45 reactive molecules . However, three of six human T lymphotropic virus type I transfected cell lines, although expressing beta 2-m free H chains, do not generate soluble molecules . Finally, human sera were found to contain considerable amounts of beta 2-m free HLA class I H chains . The average amount of these molecules in sera of individuals with one positive LA45 allele was determined to be 46.9 +/- 38.6 nM/liter.

J Cell Physiol, 1993 Sep, 156(3), 588 - 600
Apotransferrin stimulation of thyroid hormone dependent rat pituitary tumor cell growth in serum-free chemically defined medium: role of FE(III) chelation; Eby JE et al.; Triiodothyronine (T3) dependent growth of GH1 rat pituitary tumor cells in serum-free defined culture requires apotransferrin (apoTf) (Sirbasku et al.: Mol . Cell . Endocrinol., 77:C47-C55, 1991) . Diferric transferrin (2Fe.Tf) also is necessary as an iron source (Eby et al.: Anal . Biochem., 203:317-325, 1992) . Further, T3 dependence is prevented by soluble Fe(III) addition to the medium (Sato et al.: In Vitro Cell . Dev . Biol., 27A:599-602, 1991) . While our data suggested that apoTf caused growth by chelation of Fe(III), direct evidence was required . We used urea polyacrylamide gel electrophoresis along with autoradiography and Western immunoblotting to measure the Fe(III) content of growing GH1 cell cultures and identify the apoTf, mono-metal transferrins and 2Fe.Tf present . We found that apoTf per se did not cause growth but instead chelated inhibitory levels of Fe(III) . In fact, apoTf need not be present at all provided that Fe(III) is reduced to < or = 0.6 microM . In addition, other protein and non-protein Fe(III) chelators were shown to be as effective as apoTf . Here, we report that pituitary cells are completely inhibited by > or = 1.2 microM Fe(III), which are concentrations which might be expected in many culture media and usually are not thought to influence growth . The high sensitivity of pituitary cells to Fe(III) suggests further study to determine what cellular functions are affected and how they interfere with thyroid hormone dependence.

Scand J Immunol, 1993 Sep, 38(3), 267 - 72
Expression of human IL-2 receptor alpha- and beta-chains using the baculovirus expression system; Lindqvist C et al.; The genes encoding the alpha- and beta-chains of the human interleukin-2 receptor were expressed in lepidopteran insect cells using the baculovirus expression vector system . The corresponding genes were inserted under the polyhedrin promoter of the Autographa california nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line during viral infection . The recombinant receptor proteins were identified in the insect cell lysates by using protein dot blot and ELISA techniques . At 36 h post infection the corresponding proteins were clearly detected using anti-IL-2 alpha- and beta-receptor-specific antibodies . A large amount of the alpha-chain was also found in the supernatant culture media at 72 h post infection and metabolic labelling with {35S}-methionine indicated that it was proteolytically cleaved into a 32 kDa soluble form . A similar soluble or secreted form of the beta-chain was, however, not observed . Both receptor proteins were expressed on the surface of the insect cells as determined by flow cytometry analysis . Studies performed with the different IL-2 receptor forms (alpha- and beta-chains alone or in combination) in the presence or absence of rIL-2 suggest that the receptor proteins when expressed in infected insect cells are non-functional with respect to tyrosine phosphorylation.

Hum Cell, 1993 Sep, 6(3), 182 - 7
{In vitro study for hormones and growth factors dependent cell proliferation of endometrial adenocarcinoma cells}; Hata H et al.; Sex steroid hormone dependent cell proliferation and inducing growth factors of endometrial carcinoma cells were investigated using in vitro culture systems . The cell proliferation of Ishikawa cells derived from well-differentiated endometrial adenocarcinoma which possess both estrogen and progesterone receptors were stimulated by either estradiol added to culture media or EGF and TGF-alpha acting through EGF receptors . These stimulatory effects of TGF-alpha were antagonized by the anti TGF-alpha and EGF-receptor antibodies . The cell proliferations of other endometrial cancer cells were also inhibited by those antibodies . All endometrial cancer cells secrete TGF-alpha into their culture media measured by TGF-alpha ELISA methods . The expression of TGF-alpha mRNA and secretion of TGF-alpha of Ishikawa cells were induced by estradiol but not of hormone independent HEC-50 cells . Thus suggest that estradiol dependent growth factor should be TGF-alpha in human endometrial carcinoma cells.

Teratology, 1993 Sep, 48(3), 213 - 26
Chloroquine embryotoxicity in the postimplantation rat conceptus in vitro; Ambroso JL et al.; The embryotoxicity of the antimalarial drug chloroquine (CQ) was evaluated in vitro using the rat whole embryo culture system . CQ was found to be embryotoxic and dysmorphogenic when added directly to the culture media containing gestational day (GD) 10 rat conceptuses . Twenty-six-hr exposure to CQ elicited dose-related decreases in embryonic crown-rump length, protein and DNA contents and increases in the incidence of morphologically abnormal embryos . At 30 microM CQ, embryonic protein content was decreased to 67% and DNA content to 58% of control while the incidence of morphological abnormalities rose to 100% . Abnormal axial rotation, micro-ophthalmia, and selective cephalic hypoplasia were the most common developmental abnormalities observed . Visceral yolk sac (VYS) vasculature and blood pigmentation were also decreased in a dose-dependent manner, as was VYS DNA content (80% of control at 30 microM) . VYS protein content, however, showed an alternate pattern of response, decreasing to 87% of control at 10 microM CQ but increasing to 125% of control at 30 microM . Histologic evaluation revealed that the cytoplasm of the VYS endoderm epithelium was distended due to vacuolization produced by CQ exposure . In the embryo proper, CQ inhibited cranial neural tube development and altered the morphology of cranial neural crest cells . These observations document the in vitro embryotoxicity of CQ and suggest altered VYS histiotrophic nutrition as well as direct embryonic effects as possible mechanisms of CQ embryotoxicity.

Matrix, 1993 Sep, 13(5), 389 - 98
Interleukin-1 alpha and epidermal growth factor synergistically enhance the release of collagenase by periosteal connective tissue in vitro; van der Zee E et al.; The effects of recombinant human interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on the release of collagenase were studied in an in vitro model system using periosteal explants from rabbit calvariae . Following an incubation period of 72 h it was shown that IL-1 alpha in combination with EGF (IL-1 alpha + EGF) induced a synergistic increase in the amount of collagenase released by periosteal explants . This increase appeared to be at least 10-fold . Most of the enzyme was present in a latent form since the increase in enzyme activity was only detectable after activation by APMA and the molecular weight as determined in immunoblots corresponded to the latent form of this enzyme . Incubations carried out with IL-1 alpha alone resulted in a 2- to 4-fold increase of total enzyme activity, whereas the amount of collagenase in media of EGF-treated periosteal did not surpass control values . A neutralizing anti-IL-1 alpha antibody completely blocked the enhanced release of collagenase as induced both by IL-1 alpha and by IL-1 alpha + EGF . Indomethacin partially prevented the IL-1 alpha + EGF-induced increase in enzyme release, suggesting the involvement of prostaglandins . The amount of tissue inhibitor of metalloproteinases (TIMP) as determined by ELISA was slightly elevated in culture media obtained from all cytokine-treated explants . Comparable results were obtained by Western blot analysis as well as by a functional bioassay . It is suggested that the concomitant presence of the cytokines IL-1 alpha and EGF may play an important role in collagenase-mediated degradation of collagen.

Gen Comp Endocrinol, 1993 Sep, 91(3), 318 - 29
Synthesis of vitellogenin by eel (Anguilla anguilla L.) hepatocytes in primary culture: requirement of 17 beta-estradiol-priming; Peyon P et al.; Control and 17 beta-estradiol-primed eels were used to investigate the hormonal requirement of vitellogenesis in an immature fish, the eel . A primary culture of isolated liver cells from female silver eels was developed . The hepatocytes were maintained as monolayers on poly-L-lysine coated dishes for up to 12 days in a defined medium alone or supplemented with 17 beta-estradiol (E2, from 10(-8) to 10(-5) M) . The amounts of vitellogenin (Vg) in the cells and secreted into the medium were measured at 2-day intervals using a homologous vitellogenin ELISA . Different E2-priming conditions were determined before hepatocyte isolation (one injection of 250 micrograms of E2 21 days, 17 days, or 24 hr) . The vitellogenic response of hepatocytes to E2 stimulation was studied in relation to the duration of the E2-priming . After 8 days of culture, when hepatocytes from control eels were used, Vg was undetectable both in cells and in culture media, even if the culture was performed in the presence of E2 10(-5) M . However, Vg was detectable both in cells and in culture media of hepatocytes from E2-primed eels . If the priming was performed 24 hr before the culture, the Vg synthesis significantly increased (P < 0.001) in the presence of E2 10(-5) M after 10 days of culture but remained low . When the culture was performed 17 or 21 days after the priming, the level of the vitellogenic response was higher than after a short priming . In particular, with hepatocytes from 21-day E2-primed eel, the concentration of secreted Vg was 1.5 times higher than in control dishes (P < 0.01), in the presence of E2 10(-8) M after 12 days of culture . Higher doses of E2 (10(-5) M) increased Vg 2.7-fold over control values (P < 0.01) after 4 days of culture . In control dishes, cultured without steroid, the amounts of secreted and intracellular Vg remained unchanged over 12 days of culture (respectively, 72.8 +/- 2.7 ng/10(6) cells/48 hr and 28.7 +/- 2.7 ng/10(6) cells) . These results show that cultured hepatocytes retain their functional capacity by synthesizing a specific protein, Vg, in the presence of E2 and there are dose- and time-related effects of E2 on in vitro Vg synthesis . The induction of hepatic vitellogenesis in vitro requires a preliminary in vivo E2-priming.

Am J Physiol, 1993 Sep, 265(3 Pt 1), C801 - 5
Autocrine control of wound repair by insulin-like growth factor I in cultured endothelial cells; Taylor WR et al.; The repair process of the vascular endothelium is modulated by growth factors from both endogenous (within the vessel wall) and exogenous (blood borne) sources . We utilized a tissue culture model of endothelial wounding to gain further insight into the potential autocrine control of proliferation during wound repair . Cultured porcine aortic endothelial monolayers were mechanically wounded by passing a 7-mm sterile glass rod over the surface of the culture . Proliferation at the wound edge was quantified using {3H}thymidine autoradiography . In wounded cultures incubated in media supplemented with 10% fetal calf serum, 81 +/- 2% of the nuclei at the wound edge were labeled . When the cultures were incubated in serum-free media, proliferation at the wound edge was only slightly diminished with 65 +/- 3% (P < 0.05) of the cells labeled . These findings raise the possibility that there is a significant contribution from autocrine growth factors to endothelial wound repair . To evaluate the potential role of insulin-like growth factor I (IGF-I) in the wound repair process, we used a radioimmunoassay to measure IGF-I secretion . Wounded cultures exhibited a 187 +/- 58% increase in IGF-I production when compared with nonwounded cultures (P < 0.05) . To determine the extent to which endogenous IGF-I mediates the proliferative response of endothelial cell monolayers to wounding, wounded cultures were incubated with inactivating concentrations of IGF-I antibody . When IGF-I antibody was present in the culture media, only 26 +/- 3% of the nuclei at the wound edge were labeled with {3H}thymidine (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Eur Fertil, 1993 Sep-Oct, 24(5), 207 - 13
Factors affecting human blastocyst formation in vitro and freezing at the blastocyst stage; Menezo YJ et al.; Whatever the culture medium, embryo culture generally leads to a major loss of viability in mouse, rabbits even if the morphological development of the embryo is preserved . Moreover, Embryo metabolism is commonly depressed in culture media . The protein turnover is accelerated and the quality of the metabolites transport systems is impaired . Various coculture systems have been designed to avoid this loss of viability and in some animal species, to overcome the so called "embryo developmental arrest" usually observed at the approximate time of genomic activation . Moreover, it is clear now that cocultured embryos have usually higher cells numbers than those observed for embryos cultured in classical culture media . In the human, the problems seem less complicated because embryos can be transferred into the uterus on the second day post fertilization, at a time when they would normally be in the Fallopian tube: this is not possible in animal species . Also, blastocysts can be obtained, even at low rates, in conventional culture media and there is no apparent block of development . In this paper, we will present an overview of Cocultures in different species . Then, we will focus on the Human including the blastocyst formation rate and freezing at the Blastocyst stage . At the beginning of the Story, For coculturing, 2 ideas were put forward: The use of embryonic tissue (trophoblast) to help the embryo through an autocrine effect . The use of female genital tract cells, to assist the embryo through a paracrine effect.

Mol Microbiol, 1993 Sep, 9(5), 1071 - 7
Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin; Smith JM et al.; Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxin called restrictocin . To investigate the role of restrictocin in IPA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption . These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay . Western blot analysis of one disruptant, using an anti-restrictocin monoclonal antibody, confirmed that the toxin is not produced . The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media . The pathogenicity of two disruptants was assessed in a murine model of IPA . There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant . In addition, histological examination of infected lung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclear leucocyte response . Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.

Braz J Med Biol Res, 1993 Sep, 26(9), 955 - 9
Intraretinal neurotrophic activity prevents the degeneration of ganglion cells in retinal explants; Rehen SK et al.; The degeneration of ganglion cells was studied in neural retina explanted from the eyes of newborn rats . The ganglion cells were detected by the presence of retrogradely transported horseradish peroxidase injected into the superior colliculus . The time course of cell death among the axotomized ganglion cells in the explants was similar to that found in vivo after axotomy in neonatal rats . The effect of culture media conditioned with retinal cells from either newborn rats or chick embryos was tested on the survival of ganglion cells in the explants . Both conditioned media increased 2- to 3-fold the survival of rat retinal ganglion cells after 2 days in culture . The data show that soluble trophic factors released by retinae of distinct species can influence the survival of ganglion cells within their histotypic microenvironment.

Carcinogenesis, 1993 Sep, 14(9), 1815 - 9
Growth inhibition and differentiation of HT-29 cells in vitro by inositol hexaphosphate (phytic acid); Sakamoto K et al.; Inositol hexaphosphate (InsP6 or phytic acid) has been shown to have antineoplastic action in in vivo models of colon carcinogenesis . We therefore investigated its effect on proliferation and differentiation of the human colon cancer cell line HT-29 in vitro . Proliferation was evaluated by neutral red incorporation assay, and differentiation was assessed by expression of the markers, cytokeratin, carcinoembryonic antigen (CEA) and beta-D-galactose-{1-->3}-N-acetyl-galactosamine (Gal-GalNAc) . InsP6 in the culture media (0.66-10 mM) inhibited cell proliferation in a dose-dependent manner (P < 0.001), while inositol or inositol hexasulfate used as controls or media without InsP6 did not show any suppressive effect . The expression of the tumor marker, Gal-GalNac, was augmented (100.7% increase) by low dose (0.66 mM) of InsP6 but was subsequently suppressed with higher concentrations of InsP6 . The expression of cytokeratin and CEA were both augmented by either InsP6 or inositol at all concentrations tested, although the degree of augmentation was milder with inositol than with InsP6 . The combination of InsP6 and inositol (both 0.66 mm) resulted in augmentation (P < 0.001) of cytokeratin expression, while that of CEA remained unchanged . The inhibitory effect of InsP6 on cell proliferation was not altered by combination with additional inositol at any concentrations tested . Our results show that InsP6 inhibits cell proliferation and concomitantly increases differentiation and is therefore a candidate chemopreventive and chemotherapeutic agent for human large intestinal cancer.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1044 - 50
Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells; Parola M et al.; Hepatic fat-storing cells (FSC) play a key role in the development of fibrosis as a major source of collagen and other extracellular matrix (ECM) proteins in the injured liver . Both experimental and clinical studies have shown that lipid peroxidation is often associated with the development of liver fibrosis . Here we report that exposure of cultured human liver FSC to the pro-oxidant system ascorbate/iron results in an early induction of lipid peroxidation, as monitored in terms of MDA and fluorescent aldehyde/protein adducts production, and in a significant increase of the constitutive expression of procollagen type I mRNA paralleled by the accumulation of the protein in cell culture media . This fibrogenic effect is almost completely abolished by pretreatment of FSC cultures with the antioxidants alpha-tocopherol (Vitamin E) or diphenylphenylendiamine (DPPD) . Moreover, treatment of FSC with 1.0 microM 4-hydroxynonenal (HNE), a highly reactive aldehydic end-product of lipid peroxidation, results in a significant stimulation of procollagen type I gene expression and synthesis, suggesting that this aldehyde also exerts profibrogenic activity . These findings indicate that oxidative reactions can directly influence procollagen I gene expression and synthesis in FSC, thus contributing to the development of liver fibrosis.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1234 - 41
Significance of vascular endothelial growth factor/vascular permeability factor for solid tumor growth, and its inhibition by the antibody; Kondo S et al.; Angiogenesis is essential for successful tumor growth in vivo . There is a hypothesis that tumors secrete a putative tumor angiogenic factor (TAF) to facilitate blood vessel formations . Although several endothelial growth factors have been reported, it remains unclear whether these factors function as TAF in vivo . Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) is a vascular endothelial mitogen that can increase blood vessel permeability . We have established a cell line (HeLa/v5), which secretes VEGF/VPF, by transfection of human VEGF/VPF cDNA . HeLa/v5 showed higher angiogenic activity, taken/planted ratio and tumor growth rate than the control transformant (HeLa/c), when they were implanted to nude mice . Administration of a polyclonal antibody, which neutralizes the mitogenic activity of VEGF/VPF in vitro, to the tumor implanted nude mice suppressed the in vivo growth of HeLa/v5 . Furthermore, all 8 tumor cell lines we tested secrete VEGF/VPF into culture media . Our findings indicate that VEGF/VPF is a tumor angiogenic factor.

Mol Cell Biochem, 1993 Aug 11, 125(1), 19 - 25
Effects of glycated albumin on mesangial cells: evidence for a role in diabetic nephropathy; Ziyadeh FN et al.; Nonenzymatically glycated proteins are preferentially transported across the glomerular filtration barrier, and the glomerular mesangium in diabetes is bathed with serum containing increased concentrations of glycated albumin . We investigated effects of glycated albumin on mesangial cells, which are involved in diabetic nephropathy . {3H}-thymidine incorporation was significantly inhibited when murine mesangial cells were grown in culture media containing human serum that had been nonenzymatically glycated by incubation for 4 days with 28 mM glucose . This inhibition was reversed when monoclonal antibodies that selectively react with Amadori products of glycated albumin were added to the culture media . Purified glycated albumin containing Amadori adducts of the glycation reaction induced significant inhibition of thymidine incorporation and stimulation of Type IV collagen secretion compared with cells cultured in the presence of purified nonglycated albumin . These changes were prevented when monoclonal antibodies specifically reactive with fructosyl-lysine epitopes in glycated albumin were added to the cultures . The antibodies had no effect on growth or collagen production in the presence of nonglycated albumin . The results provide the first evidence directly implicating Amadori adducts in glycated albumin in the pathogenesis of diabetic nephropathy, which is characterized by decreased cellularity in association with expansion of the mesangial matrix.

Clin Infect Dis, 1993 Aug, 17 Suppl 1, S243 - 9
Extragenital Mycoplasma hominis infections in adults: emphasis on immunosuppression; Meyer RD et al.; Mycoplasma hominis, a commensal organism that is potentially pathogenic both in maternal perinatal and in neonatal infections, also causes nongenitourinary infections in adults . We reviewed the clinical features of cases from the literature and emphasized recent cases . Infection sites were classified as blood, vascular sites, wounds, central nervous system, joints, and respiratory tract . Twenty-one of 31 newly summarized cases and 32 of 67 overall cases were associated with immunosuppression and/or hypogammaglobulinemia . Clinical suspicion, use of appropriate culture media, and determinations of gamma-globulin levels and of antibodies specific to Mycoplasma are indicated in the proper clinical setting . These types of M . hominis infection appear to be linked to suppressed cell-mediated immunity and/or hypogammaglobulinemia.

Gan To Kagaku Ryoho, 1993 Aug, 20(11), 1457 - 60
{Adoptive immunotherapy in patients with multiple hepatic cancers using lymphokine activated killer cells (LAK) and interleukin-2}; Kanai T et al.; Tumor infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) were isolated from 5 patients with multiple hepatocellular carcinoma (HCC) and with metastatic liver tumor . Proliferation of lymphocytes was observed following addition of interleukin-2 (700 JRU/ml) into the culture media . After analysis of LAK cells by FACS, the antitumor activities were examined . Higher levels of cytotoxic activity against tumor cells (K 562 and Daudi) have continued during the culture period ranging from 2 to 5 weeks . Adoptive immunotherapy was performed using TIL-LAK and PBL-LAK via hepatic artery for three patients with HCC and one patient with metastatic tumor . The average number of administered lymphocytes was 1 x 10(9) . Reduction of tumor size was observed after this therapy, which caused no severe side effects exclusive of high fever and pleural effusion . Our results indicate that the treatment using lymphokine activated killer cells and interleukin-2 may be a promising modality for patients with multiple HCC.

J Clin Invest, 1993 Aug, 92(2), 671 - 8
Human keratinocytes are a major source of cutaneous platelet-derived growth factor; Ansel JC et al.; PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing . While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described . In this manuscript, we report the production of PDGF by cultured human keratinocytes . Both PDGF A and B chain mRNA can be detected in cultured cells . While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts . No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth . Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis . No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

J Neurochem, 1993 Aug, 61(2), 457 - 63
Characterization of the alterations in purine nucleotide metabolism in hypoxanthine-guanine phosphoribosyltransferase-deficient rat neuroma cell line; Zoref-Shani E et al.; A rat neuroma cell line (B103 4C), deficient of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), was utilized as a model tissue in search for the biochemical basis of the Lesch-Nyhan syndrome (LNS) . The HGPRT-deficient neurons exhibited the following properties: an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92% and 0.69% of normal, respectively); a significant increase in the availability of 5'-phosphoribosyl-1-pyrophosphate; a three- to fourfold acceleration of the rate of de novo nucleotide synthesis; a normal excretion of xanthine, but 15-fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purine nucleotide content, including the absence of 5-amino-4-imidazole carboxamide nucleotides (Z-nucleotides), but enhanced turnover of adenine nucleotides (loss of 86% of the radioactivity of the prelabeled pool in 24 h, in comparison to 73% in the normal line), and an elevated UTP content . The results suggest that, under physiological conditions, guanine salvage does not occur in the normal neurons, but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool . The finding of the normal profile of purine nucleotides in the HGPRT-deficient neurons indicates that the lack of hypoxanthine salvage is adequately compensated by the enhanced de novo nucleotide synthesis . These results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z-nucleotide accumulation, occurs in HGPRT-deficient neurons and that these are etiological factors causing the neurological abnormalities in LNS.(ABSTRACT TRUNCATED AT 250 WORDS)

Nippon Jinzo Gakkai Shi, 1993 Aug, 35(8), 887 - 91
Characterization of the glomerular endothelial cell in culture; Nitta K et al.; Because of difficulties associated with the culture, cloning and propagation of glomerular endothelial cells (GENs), the biological properties of these cells remain largely unknown . We modified the methods established by Ballermann to propagate GENs from adult bovine kidney . We found that the addition of insulin, transferrin and selenium into the standard culture media was an important step in promoting the propagation of the first clone from a single cell and in maintaining the viability of the cells . These cells expressed factor VIII-related antigen and took up acetylated-LDL, but did not contain the Weibel-Palade body, unlike endothelial cells derived from large vessels . Furthermore, GENs were compared with aortic endothelial cells (AECs) to investigate the differences in culture conditions . Compared with AECs, GENs required a higher concentration of serum and the supplementation of growth factor to maintain their biological activity . In addition, GENs were very susceptible to trypsinization and produced prostaglandin E2 as a major cyclooxygenase product, whereas AECs produced PGI2 . These findings suggest that GENs will be easily obtained from adult bovine kidney in culture and provide useful information on the functional properties of these cells under physiological and pathophysiological conditions.

Diagn Microbiol Infect Dis, 1993 Aug-Sep, 17(2), 181 - 3
Comparison of fetal bovine serum and colostrum-free bovine serum supplements of virus culture; Maggs V et al.; Incidence of virus isolation and length of time to cytopathic effect were compared in human diploid fibroblasts by using culture media supplemented with either fetal bovine serum or a less expensive alternative, colostrum-free bovine serum . The two systems were equivalent for diagnosis of 386 viral isolates.

Curr Eye Res, 1993 Aug, 12(8), 703 - 9
Transforming growth factor-beta stimulates collagen and fibronectin synthesis by human corneal stromal fibroblasts in vitro; Ohji M et al.; The effects of transforming growth factor-beta (TGF beta) and epidermal growth factor (EGF) on the synthesis of collagen and fibronectin, and on the proliferation of human corneal stromal fibroblasts in vitro, were evaluated . Human corneal stromal fibroblasts in culture were incubated for 48 hours with TGF beta or EGF in the absence of serum . Collagen and fibronectin in the culture media were measured by a collagenase-digestion assay and a competitive ELISA, respectively . The effects of the growth factors on proliferation were assessed by 3H-thymidine incorporation . Collagen synthesis was dose-dependently stimulated by TGF beta; at a concentration of 1 ng/ml of TGF beta, a 120% increase in collagen synthesis was seen over that of controls (p < 0.01) . EGF, at a concentration of 10 ng/ml, induced a 40% increase in collagen synthesis over that of controls (p < 0.01) . The maximum stimulation by TGF beta was greater than that by EGF (p < 0.05) . Fibronectin synthesis was stimulated by TGF beta and EGF in a dose-dependent manner; 230% (p < 0.001) and 210% (p < 0.01) increases in fibronectin synthesis were caused by 10 ng/ml TGF beta and EGF, respectively . TGF beta and EGF dose-dependently stimulated 3H-thymidine incorporation . The maximum increases in 3H-thymidine incorporation reached 180% (p < 0.001) and 190% (p < 0.001) over that in controls, at 10 ng/ml concentrations of TGF beta and EGF, respectively . In conclusion, both TGF beta and EGF are potent stimulants of collagen and fibronectin synthesis and proliferation . Therefore, these two growth factors may be effective alternatives or additional choices for the treatment of corneal ulcer.

Neurochem Int, 1993 Aug, 23(2), 123 - 9
Microencapsulation of genetically engineered fibroblasts secreting nerve growth factor; Maysinger D et al.; We demonstrated that genetically modified fibroblasts can be encapsulated into biocompatible, biodegradable spheres retaining their viability and capacity to continuously secrete nerve growth factor (NGF) for at least two months . Genetically engineered rat fibroblasts producing NGF were encapsulated in an alginate-polylysine gel with the ultimate objective of improving transplantation methodologies . Cultures were suspended in a sodium alginate solution and the suspension was extruded drop-wise into a solution of calcium chloride . Morphological properties of the spheres were assessed by light and electron microscopy . The spheres had a homogenous external membrane, without fibroblasts, protruding from the surface of the capsular membrane . The NGF determinations in culture media showed that encapsulated fibroblasts continued to synthesize NGF for at least 60 days . We also confirmed that secreted NGF was biologically active, by assessing the induction of choline acetyltransferase (ChAT) activity in dissociated embryonic rat septal cultures . These results encourage further studies using in vivo models to determine the value of applying microencapsulated genetically modified cells secreting trophic factors as a therapeutic strategy for central nervous system (CNS) injuries.

Cancer, 1993 Aug 1, 72(3), 931 - 7
Characterization of a CD34+ cell line established from a child with large cell cutaneous lymphoma; Lorenzana AN et al.; BACKGROUND . Lymphoma presenting with skin involvement has heterogeneous morphology and rarely is seen in children . To study the pathogenesis of this disease, lymphoma cells from a child with B-cell large cell lymphoma of the skin were cultured in vitro . METHODS . Lymphoma cells cultured on a feeder layer under hypoxic conditions grew in vitro after a latency period of 2 weeks . Since interleukin-6 (IL-6) induces final differentiation of activated B-lymphocytes, the cell line was evaluated for the presence of IL-6 receptors and biologic response to IL-6 . RESULTS . An Epstein-Barr virus (EBV)-negative cell line (UoC-B2) was established which expressed CD34, CD45, HLA-DR, CD19, CD20, sIgM, sIgD, and lambda light chain . Good general concordance was observed between the patient's lymphoma and the cell line by comparing the immunophenotype, genotype, and karyotype . The UoC-B2 cells expressed surface IgM but did not secrete IgM into the culture media even in the presence of supplemental IL-6 . CONCLUSIONS . A B-lymphoid cell line (UoC-B2) was established from a child with primary cutaneous lymphoma . The cells expressed cell surface IgM and receptors for IL-6 but supplemental IL-6 had no effect on IgM production or cell proliferation.

Clin Chim Acta, 1993 Jul 16, 216(1-2), 1 - 10
Deregulated production of interleukin-4 (IL4) in autoimmune thyroid disease assayed with a new radioimmunoassay; Hirooka Y et al.; A sensitive, reproducible and specific radioimmunoassay for human interleukin-4 (IL4) has been developed . Using 125I-labeled IL4 and polyclonal rabbit antisera raised against recombinant human IL4, a competitive inhibition assay was developed which could detect 5 pg/ml of human IL4 . Other interleukins, growth factors, hormones, peptides and lectins did not affect the assay . IL4 was measured in supernatants of culture media of stimulated human peripheral blood mononuclear cells (PBMC) . Kinetics of IL4 production in PHA-stimulated PBMC from seven normal subjects revealed that the peak levels of IL4 were seen at 24 h and then declined . Peak IL4 levels in PHA stimulation of PBMC from untreated patients with autoimmune thyroid diseases (Graves' disease and chronic thyroiditis) were significantly higher than normal controls . However, after treatment, IL4 production decreased to normal . The present study demonstrates the usefulness of quantitating human IL4 produced by PBMC and that there exists a deregulated production of IL4 in autoimmune thyroid diseases.

J Biol Chem, 1993 Jul 5, 268(19), 14215 - 23
Dietary selenium affects methylation of the wobble nucleoside in the anticodon of selenocysteine tRNA({Ser}Sec); Diamond AM et al.; We reported previously that the presence of selenium in culture media of mammalian cells influences both the steady-state levels and distributions of two tRNA isoacceptors involved in the insertion of selenocysteine into protein in response to certain UGA codons . In this study, we demonstrate an increase in the levels of these isoacceptors in rats fed a selenium-adequate diet compared to animals fed a selenium-deficient diet, as well as a shift in the relative distribution toward the tRNA which elutes later from an RPC-5 column . These effects were found to occur in a tissue-specific manner . Both selenocysteine tRNAs were isolated from rat liver, sequenced, analyzed by mass spectrometry, and shown to differ only by ribose 2'-O-methylation of 5-methylcarboxymethyluridine that occurs in the wobble position of the anticodon . This modified nucleoside has been documented previously only in yeast tRNA while the corresponding 2'-O-methylribose derivative has not been observed . The structure of these nucleosides was established by mass spectrometry and confirmed by chemical synthesis . Although the role of methylation of the wobble nucleotide is not known, the differences in elution properties from RPC-5 columns are consistent with other experimental observations indicating that a change in tRNA conformation accompanies this methylation.

J Reprod Fertil, 1993 Jul, 98(2), 349 - 56
Distribution of lysophospholipids and metabolism of platelet-activating factor in human follicular and peritoneal fluids; Lepage N et al.; The distribution of lysophosphatidylcholine, lyso-platelet-activating factor and platelet-activating factor (PAF) was studied in human plasma and in follicular and peritoneal fluid . In plasma, peritoneal and follicular fluids, 51%, 87% and 89%, respectively, of the total lipids were found in the protein fraction (the density > 1.21 fraction) . Two forms of lysophospholipids were identified in this fraction: one of high affinity and one of low affinity for albumin . The metabolism of PAF in human follicular fluid, peritoneal fluid and plasma was also investigated . PAF-acetylhydrolase activity was found in both peritoneal and follicular fluids which induced a time-dependent hydrolysis of {3H}PAF . The half-life of PAF was estimated to be 7-12 min in plasma, 15-25 min in peritoneal fluid and approximately 2 h in follicular fluid . PAF-acetylhydrolase activity in embryo culture media supplemented with 10% serum was markedly inhibited by addition of commercial serum albumin . When 25 g albumin l-1 was added, 22% of {3H}PAF was hydrolysed h-1 compared with 72% in media without albumin . The concentrations of lysophosphatidylcholine measured in plasma, in follicular and peritoneal fluids were 252, 286 and 53 mumol l-1, respectively . The distribution of these lysophospholipids and the metabolism of PAF in the female genital tract fluids reported in the present study provide evidence for the involvement of these biologically active lipid mediators in a variety of reproductive processes including sperm-egg interactions and embryonic development.

Eur J Clin Chem Clin Biochem, 1993 Jul, 31(7), 441 - 6
Development of an amplified enzyme-linked immunosorbent assay for sensitive measurement of apolipoprotein B in plasma and tissue culture media; Macri J et al.; We have developed a non-competitive sandwich enzyme-linked immunosorbent assay for the quantitation of apolipoprotein B, which utilizes the biotin-avidin amplification system . All components were optimized with respect to either concentration or incubation time . The assay uses microtitre wells as a solid phase and polyclonal, affinity-purified capture antibody . The amplification system was composed of hydroxy-succinimide biotin conjugated to an affinity-purified anti-apolipoprotein B IgG and either ExtrAvidin-alkaline phosphatase or streptavidin-alkaline phosphatase . The within-run precision (n = 10) of the apolipoprotein B control (1.39 g/l) was 3%, while the between-run precision of the assay (n = 6) for 9 consecutive assays was determined to be 8% . A sensitivity of 3 ng was attained, with a mean analytical recovery of 110% . Comparison of the assay with an established immunoturbidometric method resulted in a correlation of 0.92 using patient plasma samples (n = 19) . The use of the biotin-avidin amplification system provides the sensitivity required for measuring apolipoprotein B in tissue culture media samples and circumvents the many problems associated with the direct conjugation of enzymes to antibodies . Minimal amounts of reagents are required and the assay can be performed in 5 h, making it both economical and practical for clinical laboratories.

Cell Growth Differ, 1993 Jul, 4(7), 571 - 9
Hepatocyte growth factor/scatter factor is an autocrine factor for human normal bronchial epithelial and lung carcinoma cells; Tsao MS et al.; Hepatocyte growth factor (HGF) or scatter factor (SF) has been considered primarily as an endocrine/paracrine factor . We report here that HGF/SF mRNA was expressed by cultured human normal bronchial epithelial cells and many non-small cell lung carcinoma cell lines . Scatter activity was detected in the culture media of these cells, and this activity was inhibited by a neutralizing anti-recombinant human HGF antiserum . Immunostaining confirmed the presence of immunoreactive human HGF-like protein in the cytoplasm of these cultured cells, and in ciliated columnar epithelium of normal human bronchus/bronchioles . The met/HGF/SF receptor of these cultured cells was constitutively phosphorylated on tyrosine residues . A neutralizing anti-recombinant human HGF antiserum decreased the phosphorylation of the receptor, inhibited the proliferation of 45% of the non-small cell lung carcinoma cell lines studied, and stimulated the proliferation of normal bronchial epithelial cells . Altogether, the data demonstrate that HGF/SF and/or HGF-like protein is an autocrine factor for normal and neoplastic human bronchial epithelial cells in culture.

Mol Cell Endocrinol, 1993 Jul, 94(1), 9 - 20
Long-term culture of human pancreatic islets in an extracellular matrix: morphological and metabolic effects; Lucas-Clerc C et al.; In this experiment, various conditions for embedding cultures of human pancreatic islets in type I collagen gel were studied in an attempt to maintain the highly differentiated functions of islet cells and particularly insulin secretion over a long period of time . The islets isolated by a collagenase digestion technique were plated either on or within the collagen gel and refed with either Eagle's minimum essential medium (5.5 mM D-glucose) or RPMI 1640 medium (11 mM D-glucose) supplemented with 10% FCS and antibiotics . The comparison between the two culture media showed that embedded islets cultured in RPMI had a higher basal insulin secretion rate, survived longer than their MEM counterparts, but exhibited impaired response to an acute glucose test contrasting thus with islets cultured in MEM . The secretory behaviour of islets was also related to the different morphological modifications occurring during culture . Islets directly embedded within the collagen gel more or less maintained their spherical structure and highest secretory capacities . When overlaid with a second layer of collagen, well established monolayers of human islet cells grown on collagen underwent a gradual and complete reorganization into a three-dimensional islet-like structure with a striking reinforcement of their secretory activity . Both cultures were able to survive more than 8 weeks, thus proving the usefulness of such a new model for long-term culture . In contrast, standard cultures on culture treated plastic dishes on which islets cells rapidly established wide monolayers, exhibited a rapid and definitive decline in insulin secretion with a survival not exceeding 14 days . In the light of these different culture conditions, possible mechanisms responsible for disturbance of hormonal release and their implications for in-vitro study of isolated islets functions are discussed . In conclusion, this work is a new example of the permissive effects of collagen matrices on the establishment or maintenance of tissue-like structures in vitro, suggesting the definition of a new model for the study of human pancreatic islets in long-term culture.

Int J Artif Organs, 1993 Jul, 16(7), 557 - 60
Secretion of erythropoietin from microencapsulated rat kidney cells: preliminary results; Koo J et al.; Rat kidney epithelial cells were microencapsulated within alginate-poly(L)lysine-alginate membrane . The microencapsulated cells were incubated using a culture media containing cobalt and another without cobalt . The viability was measured by trypan blue exclusion test . Secretion of erythropoietin (EPO) was measured by radioimmunoassay (RIA) . Viability of free cells was 53% . The viability of microencapsulated cells increased to 72% after 12 days of incubation and remained at this level . Samples of the culture media were collected every 2 days for RIA . Samples within the microcapsules were collected by breaking the microcapsules open . RIA of these samples showed the following for the media containing cobalt . Between day 16 and day 32 the concentrations of EPO were 5.3 mU/ml inside and 18.3 mU/ml outside the microcapsule . The medium from the same number of free cells contained 21.2 mU/ml of EPO . Culture media without cobalt collected during the same period contained 1.8 mU/ml inside and 9.9 mU/ml outside the microcapsules . The free cell culture with this media during the same period contained 8.3 mU/ml.

Biol Reprod, 1993 Jul, 49(1), 74 - 81
Effect of bovine serum albumin concentration and source on sperm capacitation in the golden hamster; Stewart-Savage J; Since virtually all experiments on sperm capacitation routinely include serum albumin in the culture media, the relative effect of BSA concentration on sperm capacitation was investigated . The fertile life of hamster sperm, as measured by the ability to penetrate the zona pellucida, decreased from 5 to 3 h as the concentration of BSA was increased from 3 to 18 mg/ml . To determine the extent of sperm capacitation in sperm populations that resulted in 100% penetration, the rate and timing of sperm penetration through the zona pellucida was determined . For each insemination, the rate of sperm penetration (%/min), the time when 50% of the eggs were zona-penetrated (T50), and the sperm capacitation index (T50/rate) were calculated . The extent of sperm capacitation as measured by all three parameters increased as the duration of preincubation or the BSA concentration was increased . The data on the duration of preincubation and the BSA concentration matrix indicated two properties of the effect of BSA on sperm capacitation: 1) the extent of sperm capacitation after the first hour of preincubation was independent of BSA concentration; and 2) when sperm were preincubated for two or more hours, the capacitating effectiveness of BSA was saturable; the saturating concentration of BSA decreased as the duration of preincubation increased . To determine whether the first hour of preincubation is independent of BSA, sperm were preincubated for 1 h without BSA and then 2 h with BSA . The extent of sperm capacitation of these sperm was equal to that of the 3-h control sperm; thus the first hour of capacitation is independent of BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1993 Jul, 12(7), 2645 - 53
Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal; Skoda RC et al.; The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily . We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4 . Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl . The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl . A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal . In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells . Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells.

Arch Biochem Biophys, 1993 Jul, 304(1), 110 - 6
Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins); Hiraiwa M et al.; Prosaposin contains separate domains in tandem for four saposins, A, B, C, and D . These mature saposins are produced by limited proteolysis of prosaposin . They are involved in lysosomal hydrolysis of GM1 ganglioside, gluco- and galactocerebrosides, sulfatides, and sphingomyelin and other sphingolipids . Prosaposin also exists as a secretory protein in body fluids . In this investigation prosaposin was expressed in Spodoptera frugiperda cells (Sf9) by infection with baculovirus containing a full length cDNA coding for human prosaposin . Prosaposin was isolated and purified from spent culture medium of the recombinant Sf9 cell cultures as well as from human seminal plasma and milk . From sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of both native human prosaposins is estimated to be 66 kDa and that of recombinant prosaposin as 58 kDa . Deglycosylation of native and recombinant prosaposins yielded a protein with a molecular weight of 54 kDa and isoelectric point of 5.4 . The N-terminal sequence of both native and recombinant prosaposins was identical (G-P-V-L-L-G-L-K) . Like mature saposins, all prosaposins possessed stimulative activity for cerebroside beta-glucosidase (saposins A and C activity), GM1 ganglioside beta-galactosidase (saposin B activity), and sphingomyelinase (saposin D activity) but not sulfatide sulfatase (saposin B activity) . Partially proteolyzed products derived from prosaposins were isolated and identified . From seminal plasma, two proteins of 48 and 29 kDa and from Sf9 culture media, two proteins of 39 and 26 kDa were characterized . N-terminal amino acid sequencing and Western blot analysis of each protein indicated that the 39-and 48-kDa proteins are cleavage products containing domains for saposins B, C, and D (trisaposins), and the 26- and 29-kDa proteins are cleavage products containing domains for saposins C and D (disaposin) . These observations suggest that proteolysis of prosaposin in these tissues occurs sequentially from the N-terminal region . Proteins involved in the initial proteolysis of prosaposin were partially characterized in human testis.

J Lab Clin Med, 1993 Jul, 122(1), 69 - 79
Cathepsin G--induced release of PAI-1 in the culture medium of endothelial cells: a new thrombogenic role for polymorphonuclear leukocytes?
Pintucci G, Iacoviello L, Castelli MP, Amore C, Evangelista V, Cerletti C, Donati MB.
Activated polymorphonuclear leukocytes (PMNs) may affect the integrity of blood vessels by endothelial cell injury . We investigated the effects of cathepsin G purified from human neutrophils on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVECs) . Cathepsin G (5 and 10 micrograms/ml) induced marked intercellular gap formation after 1 hour of treatment, whereas 1 microgram/ml did not, even after 6 hours incubation . In contrast, plasminogen activator inhibitor-1 (PAI-1) antigen levels, measured by a double antibody enzyme-linked immunosorbent assay, were significantly increased in culture media (CM) on cathepsin G (1 microgram/ml) treatment after 15 minutes (5.1 +/- 1.2 ng/ml vs 2.6 +/- 0.6 ng/ml for controls, p < 0.01) and 6 hours of incubation (69.6 +/- 17.5 ng/ml vs 40.0 +/- 9.0 ng/ml for controls, p < 0.01) . Likewise, PAI activity, measured by reverse fibrin autography, increased on cell treatment with cathepsin G . Preincubation of cathepsin G with eglin C (10 micrograms/ml) almost completely abolished the increase in both PAI antigen and activity levels induced by cathepsin G . Cycloheximide, a protein synthesis inhibitor, did not block cathepsin G-induced PAI-1 release . PAI-1 mRNA levels were not affected by HUVEC treatment with cathepsin G (1 microgram/ml for 15 minutes), even after 24 hours . In the extracellular matrix (ECM) PAI-1 antigen levels decreased to 77% and 40% of controls, respectively, after 15 minutes and 6 hours of cathepsin G (1 micrograms/ml) treatment . Reverse fibrin autography also demonstrated a dose-dependent reduction of PAI activity in the ECM on 6 hours of cell treatment with 1 or 5 micrograms/ml cathepsin G . Moreover, ECM prepared from confluent HUVECs released PAI-1 in supernatants on 1 micrograms/ml cathepsin G incubation in a cell-free system . Tissue-type plasminogen activator (t-PA) activity was strongly depressed on cathepsin G treatment, both in CM from HUVECs or in a cell-free system . Finally, PAI-1 was also released from cathepsin G-stimulated platelets in a dose-dependent manner . In summary, our results support a potentially thrombogenic role of cathepsin G, which could impair the fibrinolytic potential of the endothelium . These data give a new insight into the mechanisms by which activated PMNs may promote thrombus formation . On the other hand, the decrease of PAI-1 in ECM could favor penetration and migration of inflammatory or tumor cells through the subendothelial layers.

Bone, 1993 Jul-Aug, 14(4), 661 - 6
The culture of human osteoblasts upon bone graft substitutes; Begley CT et al.; In order to assess the ability of six potential bone graft substitutes to support the growth of human osteoblasts, these cells were grown in culture and then plated onto fragments of the six materials and cultured for a further period of 15 days . Tests to confirm the osteoblastic phenotype of the cells included spectrophotometric alkaline phosphatase assay; Western blotting of secreted osteocalcin, osteonectin, bone sialoprotein, and collagen type I; and mineralization within the cultures provided with a supplemented medium . Cells were seeded onto the materials in 24-well plates (Nunc, Naperville, IL) at density levels 12,500 cells/cm2 and 25,000 cells/cm2 . Specimens were examined after the 15-day culture period by scanning electron microscopy . At a seeding density of 12,500 cells/cm2 results showed that the human osteoblasts had greatest affinities for demineralized rat bone and demineralized Surgibone, whilst few osteoblasts were found attached to Pyrost, Surgibone, or coral . The collagen matrix of Callopat hydrated in the culture media exposing the hydroxyapatite crystals within it, and these became the foci for cell attachment and growth . At a seeding density of 25,000 cells/cm2 the osteoblasts had attached to and proliferated upon the surfaces of all the materials, forming multilayers, with the exception of Surgibone . These experiments demonstrated that all the materials, with the exception of nondemineralized Surgibone, were biocompatible for human osteoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Australas Biotechnol, 1993 Jul-Aug, 3(4), 214 - 5, 218-22
Biotechnology in India; Ghosh PK; Conventional biotechnology based industries are already established economic activities in India . Facilities have also been set up for the production of restriction endonucleases, oligonucleotides, culture media, plastic wares, micro pipettes etc., However recombinant biotechnology products are still at the research stage and market demand is met through imports . The bulk of the R & D resources for product development are currently being provided by the Government, which has also taken steps to develop trained man power, set up the infrastructure for research, and has organised demonstration projects for faster dissemination of research results to industry . The consumption of biotech products will rise from Rs . 18.74 billion in 1992 to Rs . 55.47 billion in 2000 and the expected additional investment is anticipated to be Rs . 14 billion during the next 8 years.

Nihon Kyobu Shikkan Gakkai Zasshi, 1993 Jul, 31(7), 853 - 8
{Cell-associated interleukin 1 production of alveolar macrophages from bleomycin-induced pulmonary fibrosis in rats}; Asada K et al.; In order to clarify the mechanisms of pulmonary fibrosis, we produced bleomycin (BLM)-induced pulmonary fibrosis in rats and examined the ability of alveolar macrophages (AM) to produce interleukin-1 (IL-1) . BLM (0.9 mg) was administered once via the trachea to male Wistar rats weighing about 200 g . Bronchoalveolar lavage was performed at 1, 3, 6, 9 and 12 days after administration . AM were incubated for 24 hours, then extracellular IL-1 in the supernatants and cell-associated IL-1 of AM were measured by proliferation assay of mouse thymocytes . Cell-associated IL-1 activity was measured after fixation by paraformaldehyde (PFA) . Extracellular IL-1 was detected in the culture media of AM at only day 1 after administration . On the other hand, cell-associated IL-1 was detected in AM fixed by PFA on days 1, 3, 6 and 9 after administration . AM from BLM-induced pulmonary fibrosis in rats were fixed by PFA and then were treated with anti-IL-1 alpha antibody or anti-IL-1 beta antibody . Cell-associated IL-1 activity was neutralized by treatment with anti-IL-1 alpha antibody and was not neutralized by treatment with anti-IL-1 beta antibody . Following this, the effect of cell-associated IL-1 on pulmonary fibroblasts was examined . This was estimated by the proliferation of pulmonary fibroblasts using incorporation of 3H-thymidine . When pulmonary fibroblasts were incubated with AM fixed by PFA from BLM-induced pulmonary fibrosis in rats, proliferation of pulmonary fibroblasts was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation, 1993 Jul, 56(1), 148 - 54
Paradoxical release of insulin by adult pig islets in vitro . Recovery after culture in a defined tissue culture medium; Davalli AM et al.; In this study, in vitro responsiveness to glucose of fresh and cultured islets from adult pigs was tested under both static (incubation) and dynamic (perifusion) conditions . Islets were isolated by an automated method from pancreases of 24-month-old animals and cultured overnight in CMRL 1066 and 10% FCS plus antibiotics; islets, perifused immediately after the overnight culture, showed a paradoxical decrease in insulin release when exposed to an acute glucose stimulus (16.7 mmol/L), and a normal response to acute glucose when isobutylmethylxanthine (IBMX) was added to the perifusing buffer . In addition, an acute reduction of glucose concentration in the perifusate elicited a paradoxical insulin release . At the microscope, islets appeared loose and irregularly shaped after the overnight culture; immunohistochemistry showed loss of peripheral A and other mantle cells . After the overnight culture, islets were divided into 5 groups and were cultured for a further 48 hr in different tissue culture media: CMRL 1066; RPMI 1640 (without glucose); RPMI 1640 (plus 11.1 mmol/L glucose); Ham's F12; and medium 199 (all media were supplemented with 10% FCS and antibiotics) . During this period, insulin release was 11.4 +/- 1.1 pg/islet/min in islets cultured in CMRL 1066, 16.2 +/- 2.4 in islets cultured in RPMI 1640 (11.1 mmol/L glucose), 1.8 +/- 0.2 (P < 0.001 vs . all the other groups), and 9.0 +/- 0.6 and 8.4 +/- 0.9 pg/islet/min in islets cultured in RPMI 1640 (without glucose), Ham's F12, and medium 199, respectively . After the 48-hr culture in different media, the islets' responsiveness to an acute glucose stimulus (16.7 mmol/L; static incubation) was evaluated: islets cultured in CMRL 1066 and in RPMI 1640 (with and without glucose) showed no insulin response to the acute glucose stimulus; in contrast, insulin release rose from 0.42 +/- 0.06 to 0.60 +/- 0.12 pg/islet/min (NS) in islets cultured in Ham's F12, and from 0.24 +/- 0.06 to 0.48 +/- 0.06 pg/islet/min (P < 0.001) in islets cultured in medium 199 . During perifusions, the paradoxical insulin release in response to an acute fall in glucose concentration disappeared, but a significant increase in response to high (16.7 mmol/L) glucose was observed only in islets previously cultured in medium 199 . To assess the possible role of glucagon and of cAMP, additional perifusions were done in islets cultured for 48 hr in CMRL 1066 in the presence of glucagon (10 mumol/L) and IBMX (10 mumol/L); glucagon and IBMX were unable to modify the insulin response to 16.7 mmol/L glucose.(ABSTRACT TRUNCATED AT 400 WORDS)

J Immunol Methods, 1993 Jun 18, 162(2), 193 - 200
Enzyme-linked immunosorbent assays for plasminogen activators; Buessecker F et al.; ELISA procedures are described for the quantitative analysis of the urokinase-type plasminogen activator (uPA) and of the tissue type PA (tPA) . The assays were developed to detect the respective type of PA in cell culture supernatants . TPA can be present as a single-chain or a two-chain protein; uPA as pro-uPA, high or low molecular weight uPA, respectively . In addition, both PAs can be complexed with the plasminogen activator inhibitors PAI-I or PAI-2 . Monoclonal antibodies specific for uPA or tPA were selected that recognized the distinct molecular forms of the PAs, even in the presence of fetal calf serum, which is a common--relatively ill-defined--ingredient of cell culture media . The test systems were found to be reliable, easy to perform, and to permit the detection of both types of PA in serum-free and serum-containing cell culture supernatants . Finally, the ELISA--in combination with functional tests--were used to analyse the different PA components in culture supernatants of uPA- or tPA-producing cell lines.

Biochem Biophys Res Commun, 1993 Jun 15, 193(2), 794 - 800
High glucose causes delayed fetal lung maturation as measured by fluorescence anisotropy; Gewolb IH et al.; Fluorescence anisotropy has been used to estimate the microviscosity of the surfactant phospholipid bilayer and no predict fetal lung maturity in human amniotic fluid; its usefulness in in vitro systems has been recently demonstrated . To investigate the effect of high glucose on lung development, anisotropy measurements were performed on 20-day fetal rat lung explant homogenates and culture media after culture for 48 hours in medium containing final concentrations of 10, 50, and 100mM glucose . Anisotropy of lung tissue cultured in 100mM glucose was significantly increased when compared to those cultured in 10mM glucose (p < .01) . After 48 hours, the media from samples grown in 100mM glucose had significantly higher anisotropy (.2210 +/- .0031) than did media from explants grown in 50mM glucose (.2027 +/- .0079; p < .05), or in 10mM glucose (.1886 +/- .0046; p < .001) . Relative fluorescence intensity of explants grown in 100mM glucose was 74.4 +/- 5.7% of those grown in 10mM glucose (p < .01) . Fluorescence intensity of media was also decreased by 15-30% under higher glucose considerations (p < .05) . These data suggest that surfactant synthesized and secreted under high glucose conditions, such as exist in the infant of the diabetic gestation, may have qualitative as well as quantitative changes.

Arthritis Rheum, 1993 Jun, 36(6), 772 - 80
Stimulation of the secretion of latent cysteine proteinase activity by tumor necrosis factor alpha and interleukin-1; Huet G et al.; OBJECTIVE . Cultured synovial fibroblast-like cells from 3 patients with rheumatoid arthritis (RA) and 3 patients with osteoarthritis (OA) were evaluated for their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1) . METHODS . Culture media and cell lysates were analyzed before and after high performance liquid chromatography (HPLC) using the enzymatic substrate, Z-Phe-Arg-AMC, and by immunoblotting with anti-cathepsin B antiserum . Immunolocalization of cathepsin B was studied on cell monolayers . RESULTS . Latent cysteine proteinase activity was found to be secreted spontaneously by cultured synovial fibroblast-like cells . This activity was increased after treatment with either TNF alpha or IL-1 . Stimulated protease activity was eluted by HPLC at a peak coincident with that of purified cathepsin B . By immunoblot, cell supernatants contained a 43-kd form of cathepsin B, while cell lysates contained a 30-kd form, consistent, respectively, with cathepsin B before and after cleavage of its propeptide . An intracellular increase in cathepsin B after treatment with TNF alpha was also seen with immunohistochemical studies . CONCLUSION . TNF alpha (in the 6 cases studied) and IL-1 (in 4 cases) stimulated the secretion of a latent cysteine proteinase activity from synovial fibroblast-like cells, which appears to represent primarily cathepsin B.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5191 - 3
Release of amyloid beta-protein precursor derivatives by electrical depolarization of rat hippocampal slices; Nitsch RM et al.; Proteolytic processing of the beta-amyloid protein precursor (APP) is regulated by cell-surface receptors . To determine whether neurotransmitter release in response to neuronal activation regulates APP processing in brain, we electrically depolarized superfused rat hippocampal slices and measured soluble APP derivatives released into the superfusate . Electrical depolarization caused a rapid increase in the release of both neurotransmitters and amino-terminal APP cleavage products . These derivatives lacked the APP carboxyl terminus and were similar to those found in both cell culture media and human cerebrospinal fluid . Superfusate proteins including lactate dehydrogenase were not changed by electrical depolarization . The release of amino-terminal APP derivatives increased with increasing stimulation frequencies from 0 to 30 Hz . The increased release was inhibited by the sodium-channel antagonist tetrodotoxin, suggesting that action-potential formation mediates the release of large amino-terminal APP derivatives . These results suggest that neuronal activity regulates APP processing in the mammalian brain.

Endocrinology, 1993 Jun, 132(6), 2279 - 86
Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving; Krishnan AV et al.; In studies to determine whether Saccharomyces cerevisiae produced estrogens, the organism was grown in culture media prepared using distilled water autoclaved in polycarbonate flasks . The yeast-conditioned media showed the presence of a substance that competed with {3H}estradiol for binding to estrogen receptors (ER) from rat uterus . However, it soon became clear that the estrogenic substance in the conditioned media was not a product of the yeast grown in culture, but was leached out of the polycarbonate flasks during the autoclaving procedure . {3H}Estradiol displacement activity was monitored by ER RRA, and the active substance was purified from autoclaved medium using a series of HPLC steps . The final purified product was identified as bisphenol-A (BPA) by nuclear magnetic resonance spectroscopy and mass spectrometry . BPA could also be identified in distilled water autoclaved in polycarbonate flasks without the requirement of either the organism or the constituents of the culture medium . Authentic BPA was active in competitive RRAs, demonstrating an affinity approximately 1:2000 that of estradiol for ER . In functional assays, BPA (10-25 nM) induced progesterone receptors in cultured human mammary cancer cells (MCF-7) at a potency of approximately 1:5000 compared to that of estradiol . The BPA effect on PR induction was blocked by tamoxifen . In addition, BPA (25 nM) increased the rate of proliferation of MCF-7 cells assessed by {3H}thymidine incorporation . Thus, BPA exhibited estrogenic activity by both RRA and two functional bioresponse assays . Finally, MCF-7 cells grown in media prepared with water autoclaved in polycarbonate exhibited higher progesterone receptor levels than cells.grown in media prepared with water autoclaved in glass, suggesting an estrogenic effect of the water autoclaved in polycarbonate . Our findings raise the possibility that unsuspected estrogenic activity in the form of BPA may have an impact on experiments employing media autoclaved in polycarbonate flasks . It remains to be determined whether BPA derived from consumer products manufactured from polycarbonate could significantly contribute to the pool of estrogenic substances in the environment.

Am Rev Respir Dis, 1993 Jun, 147(6 Pt 1), 1401 - 6
Detection of mycobacterial antigens present in short-term culture media using particle counting immunoassay; Drowart A et al.; Particle counting immunoassay (PACIA) was compared with the BACTEC system for detecting mycobacterial growth after short-term culture and was used to identify M . tuberculosis . The latex particles were coated with polyclonal anti-BCG or with specific 2A1-2 monoclonal antibodies . Bottles containing nonradioactive Middlebrook 7H9 liquid medium and BACTEC 12B vials were inoculated with equal amounts of mycobacteria from four reference strains (M . tuberculosis, M . kansasii, M . avium, and M . xenopi) . Using anti-BCG, PACIA detected mycobacterial antigens 3 to 6 days before the BACTEC system . M . tuberculosis was differentiated from the other mycobacteria using 2A1-2 . Seventeen clinical samples were also studied . In the same 10, the two techniques detected mycobacteria, PACIA with anti-BCG after 9 days and BACTEC 1 to 5 days later . For 9 of the 10 samples, PACIA with 2A1-2 detected M . tuberculosis after 20 days, a result confirmed with the AccuProbe system . M . xenopi was biochemically identified in Specimen 10 . Nonmycobacterial diseases were diagnosed in the 7 remaining unreactive specimens . We conclude that PACIA detects mycobacterial growth earlier than BACTEC and that M . tuberculosis can be distinguished from other mycobacteria in PACIA performed with specific monoclonal antibodies.

Virology, 1993 Jun, 194(2), 705 - 14
Characterization of avian reovirus-induced cell fusion: the role of viral structural proteins; Ni Y et al.; Cell fusion induced by avian reovirus was analyzed using virus strain FC and Vero cells . One-step growth curves showed that cell fusion was directly associated with viral replication . Cell fusion occurred most efficiently at basic pH (8.0-8.5) and fusion from without could not be demonstrated . Actinomycin D, at low concentrations, increased cell fusion, and cycloheximide prevented cell fusion, indicating that viral protein(s) were responsible for the induction of cell fusion . Immunofluorescence tests indicated that viral proteins were present on the infected cell surface . Radioimmuno-precipitation identified structural proteins mu 2C and sigma 2 as predominant viral protein species present on the infected cell surface . Cell fusion was inhibited by virus-specific antisera, suggesting that mu 2C and/or sigma 2 present on the infected cell surface were involved in the induction of cell fusion . Trypsin and chymotrypsin treatment of purified viruses cleaved both mu 2C and sigma 2 proteins, but generated different cleavage products with each protein . The addition of trypsin to the culture media following infection increased cell fusion, whereas chymotrypsin treatment decreased cell fusion . The opposite effects of trypsin and chymotrypsin on the cell fusion, together with the different specificities of these two proteases in cleavage of mu 2C and sigma 2 proteins, further suggest that the cell surface-associated mu 2C and/or sigma 2 are involved in the syncytium formation.

J Clin Endocrinol Metab, 1993 Jun, 76(6), 1483 - 8
Immunological characterization and immunocytochemical localization of an oviduct-specific glycoprotein in the human; Rapisarda JJ et al.; The objective of the current study was to generate a polyclonal antibody toward a previously described 110- to 130-kilodalton (kDa) human oviductal glycoprotein and to use the antibody to detect the protein in tissue sections, tissue culture media, and oviductal flushings . The polyclonal antibody was generated in male rabbits against the 110- to 130-kDa glycoprotein partially purified from hydrosalpinx fluid . Segments of human oviducts were either cut into 2- to 3-mm pieces and cultured for 24 h, or fixed and embedded in Araldite for light and electron microscopic immunocytochemistry . The protein was only present in midcycle oviductal flushings and was most evident in culture medium samples obtained at midcycle when analyzed on Western blots . No cross-reactivity was observed with proteins in human serum or human endometrial and cervical explant culture media . Immunoperoxidase staining was observed in the apical granules of the secretory cells lining the oviductal lumen . No staining was noted in other parts of the oviduct or in sections of human endometrium and cervix . Indirect immunogold localization demonstrated specific clustering of gold particles over the apical granules of the secretory cells . In summary, a polyclonal antibody to a 110- to 130-kDa human oviductal glycoprotein was successfully generated . This protein is found in the secretory cells and is released into the oviductal lumen . The synthesis of this protein appears to require elevated levels of estrogen and may play a role in early reproductive events occurring within the oviduct.

J Immunol, 1993 Jun 1, 150(11), 5094 - 103
Cytokine mediation of the suppressive effect of ferritin on colony-stimulating factor-1-dependent monocytopoiesis; Kreisberg R et al.; Peptide-specific IgG from a rabbit immunized with an alanine-lysine-proline-arginine ((ALA1)-tuftsin) containing 14-mer "ferritin" peptide neutralized rat liver ferritin inhibition of in vitro CSF-1-dependent monocytopoiesis . Antiferritin IgG similarly neutralized the inhibitory effect of ferritin but did not neutralize peptide inhibition of the in vitro myelopoietic response . No cross-reactivity between the respective antibodies and Ag was detected either by Western immunoblot or by competitive ELISA . Depletion of adherent cells before marrow cell culture significantly reduced the inhibitory effect of ferritin but did not influence peptide inhibition of CSF-1-stimulated colony formation . Adherent marrow cells and P388D1 cells treated with both CSF-1 and ferritin, but not either alone, produced inhibitory supernatant culture media that were neutralized by antipeptide but not antiferritin IgG . High resolution molecular sieve chromatography of the inhibitory adherent marrow cell and P388D1 supernatants resolved two peaks of 50 to 60 kDa and approximately 30 kDa in each . The inhibitory activity in all four peaks was neutralized by antipeptide but not antiferritin IgG . The ferritin/CSF inhibitors were not further characterized although identity with IL-6, IL-8, TNF-alpha, transforming growth factor-beta, and IFN-alpha/beta could be eliminated . The results indicate that ferritin inhibition of CSF-1-dependent monocytopoiesis is mediated by an endogenously produced inhibitor, or inhibitors, that shares antigenic similarity with the (ALA1)-tuftsin-containing 14-mer peptide and that adherent marrow cells, most likely monocytes or macrophages, produce the endogenous inhibitors in response to both CSF-1 and ferritin.

J Nutr, 1993 Jun, 123(6), 1003 - 9
Zinc protects against tumor necrosis factor-induced disruption of porcine endothelial cell monolayer integrity; Hennig B et al.; Some nutrients influence the metabolic response of cytokines such as tumor necrosis factor . Inadequate levels of the essential trace element zinc may play a role in tumor necrosis factor-induced disruption of the vascular endothelial barrier function . To test this hypothesis, endothelial cells cultured on polycarbonate filters or culture plates were exposed to six different treatments for 3 d: medium 199 enriched with 5% fetal bovine serum (control), control+two levels of supplemental zinc (7.7 and 12.3 mumol/L medium), tumor necrosis factor (5 x 10(5) U/L) and tumor necrosis factor+the two levels of Zn as noted previously . Endothelial barrier function, expressed as albumin transfer across cultured endothelial monolayers, was not affected by Zn enrichment alone . Tumor necrosis factor treatment significantly increased albumin transfer compared with control cultures . The lower concentration of Zn partially and the higher concentration totally prevented the tumor necrosis factor-induced increase in albumin transfer . The increase in cytosolic release of {3H}adenine (marker of cell injury) induced by tumor necrosis factor was prevented by added Zn . Tumor necrosis factor treatment significantly decreased angiotensin-converting enzyme activity, and tumor necrosis factor also decreased activities of two other membrane-bound enzymes, total ATPase and Ca(2+)-ATPase . These activities all were restored by Zn enrichment . Tumor necrosis factor treatment caused a decrease in cellular Zn concentration, which was prevented when the culture media were enriched with Zn . These data suggest that an important relationship exists between Zn status and tumor necrosis factor-induced endothelial cell dysfunction.

Int J Dev Neurosci, 1993 Jun, 11(3), 347 - 55
Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells; Colombo JA et al.; Normal human (week 17-20) and rat (E16-17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions . Phase-bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi-automated procedures . Subcultured astroglia were immuno-reacted against glial fibrillary acidic protein and fibronectin . Rat and human amniotic fluid allowed survival and growth of neuronal and non-neuronal cells . Human amniotic fluid samples were trophic in variable degrees . Cerebral cortex subcultured astroglia usually expressed a radial-like morphotype . Although charcoal-adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment . Growth of cell processes in neuronal- and glial-like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors . It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons . Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid . Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum . The role of amniotic fluid during the early phases of brain organogenesis is discussed.

Korean J Parasitol, 1993 Jun, 31(2), 117 - 27
{Proteinase activity in the isolates of Trichomonas vaginalis according to their pathogenicity}; Shim YK et al.; Ten axenic isolates of Trichomonas vaginalis were subcutaneously injected to the BALB/c mice in order to assess their pathogenicity by means of so-called "mouse assay" method . All the isolates revealed neutral and acid proteinase activities both in their lysates and in culture media, but the specific activities of both proteinases in the severely pathogenic group were significantly higher than the mildly pathogenic group (p < 0.05) . In the SDS-PAGE system in which the electrophoretic gels contained 0.4% gelatin as the substrate, five different banding patterns of trichomonal proteinases were detected, and the patterns were closely related with the pathogenicity of the isolates of T . vaginalis . All five bands might be regarded as cysteine proteinases group in the inhibitor assays . The cytotoxicity of the lysates of T . vaginalis to the target Chinese hamster ovarian (CHO) cell line was also significantly different according to the pathogenicity of the isolates, and generally lower in the lysates treated with cysteine proteinase inhibitors than in the control lysates . In summarizing the results, it might be considered that the proteinases of T . vaginalis showing five electrophoretic banding patterns are closely related with the pathogenicity and cytotoxicity of the isolates of T . vaginalis.

Hum Gene Ther, 1993 Jun, 4(3), 291 - 301
Expression of human factor IX by microencapsulated recombinant fibroblasts; Liu HW et al.; Deficiency of clotting factor IX (FIX) causes hemophilia B in humans . We propose a novel approach to its treatment by engineering FIX-secreting cell lines suitable for implantation in different allogeneic hosts . To prevent graft rejection following implantation, the recombinant cells can be protected with biocompatible membranes that permit exit of FIX but not entry of cellular immune mediators . To explore the feasibility of this approach, we now report on the creation of mouse Ltk- fibroblast cell lines that can deliver FIX through such immune-protective membranes . Mouse fibroblasts (Ltk-) were transfected with the cDNA for human FIX and clones secreting high levels of FIX were isolated . About 70% of the secreted FIX was biologically active . Over 98% of the recovered biological activity was precipitable by barium citrate, indicating appropriate . gamma-carboxylation of the secreted FIX . The secreted FIX was similar in molecular weight and immunoreactivity to plasma-derived human FIX . Upon enclosure in microcapsules fabricated from the biocompatible polymers, alginate-polylysine-alginate, the cells survived the encapsulation procedure with about 70-90% viability, proliferated within the microcapsules to twice their original number within 2 weeks, and continued to secrete FIX into the culture medium at similar rates as the unencapsulated cells . The biological activity, degree of post-translational gamma-carboxylation, and immunoreactivity of the FIX recovered from the culture media of the encapsulated cells were identical to those of the FIX secreted by the unencapsulated cells . In conclusion, fibroblasts engineered to secrete recombinant human FIX can proliferate and continue to secrete biologically active FIX through the alginate microcapsules . This demonstrates the feasibility of using microencapsulated recombinant cells to deliver human FIX and the potential for allogeneic somatic gene therapy for hemophilia B.

Biol Reprod, 1993 Jun, 48(6), 1334 - 40
Polypeptides synthesized and released by rat ectopic uterine implants differ from those of the uterus in culture; Sharpe KL et al.; Unique endometriosis-specific secretory proteins would be of paramount importance as noninvasive markers for diagnosis and evaluation of therapeutic approaches for endometriosis . Furthermore, identification of endometriosis-specific secretory proteins may be an important step towards understanding the pathophysiology of endometriosis-associated pain and infertility . Therefore this study was designed to assess protein synthesis and secretion by ectopic uterine implants from steroid-treated and reproductively cyclic rats with surgically induced endometriosis . Uteri, ectopic uterine implants, and control tissues were incubated in L-{35S}methionine or D-{6-3H}glucosamine for 0-24 h and 24-48 h . De novo-synthesized proteins released into the culture media were identified using two-dimensional SDS-PAGE, fluorography, and computer-assisted image analysis . Two distinct groups of ectopic uterine implant proteins were identified: ENDO I (M(r) 40,000-50,000; pI 4.0-5.2) and ENDO II (M(r) 28,000-32,000; pI 7.5-9.0) were produced by ectopic uterine implants and not the uteri . A third group of proteins, previously identified in culture media of the uteri from progesterone-treated rats and called PUP-1 (M(r) 70,000; pI 5.7), was synthesized and secreted by ectopic uterine implants 24-48 h later than in parallel uterine cultures . The detection of ectopic uterine implant proteins suggests biochemical characteristics of the ectopic tissue that may be used to develop unique markers for endometriosis . Furthermore, the delayed synthesis and secretion of the uterine protein PUP-1 by the ectopic uterine implants illustrates yet another example of the asynchronous behavior of these two tissues, which may be related to the etiology or pathophysiology of the disease.

J Bone Joint Surg Am, 1993 Jun, 75(6), 835 - 44
Fibroblast response to metallic debris in vitro . Enzyme induction cell proliferation, and toxicity; Maloney WJ et al.; Bovine synovial fibroblasts in primary monolayer culture were exposed to particulate metallic debris . The effects of the metallic particles on the synthesis and secretion of proteolytic enzymes and on cell proliferation and viability were examined . Uniform suspensions of titanium, titanium-aluminum, cobalt, and chromium particles, ranging in size from approximately 0.1 to ten micrometers (average, one to three micrometers), were prepared; the particle concentrations (the volume of particles divided by the total volume of the suspension) ranged from 0.0005 to 5 per cent . Aliquots of the particle suspensions were added to the synovial fibroblast cultures . The final particle concentrations in the media ranged from 0.0000083 to 0.83 per cent . After seventy-two hours of exposure, each medium was harvested and was assayed for proteolytic and collagenolytic activity and for hexosaminidase levels . Neutral metalloproteases, quantified by collagenolytic and caseinolytic (proteolytic) activity, represent enzymes, secreted by cells, that are capable of degrading extracellular matrix . Hexosaminidase is a marker for lysosomal enzyme activity that can include more than thirty enzymes, such as proteases, lipases, nucleases, and phosphatases . Cell proliferation was quantified by uptake of 3H-thymidine . Cell morphology was examined by scanning electron microscopy . Titanium, titanium-aluminum, and chromium significantly stimulated 3H-thymidine uptake at low particle concentrations (p < 0.01, p < 0.002, and p < 0.002, respectively) . Exposure to cobalt, even at the lowest particle concentration, resulted in a significant decrease in thymidine uptake (p = 0.027) . At the highest particle concentrations, all particles were toxic, as evidenced by the absence of thymidine uptake . At high particle concentrations, all of the metals caused a decrease in caseinolytic (proteolytic) and collagenolytic activity in the culture media . Titanium elevated the lysosomal enzyme marker, hexosaminidase, except at high concentrations . Chromium and titanium-aluminum had no significant effect on hexosaminidase at any particle concentration, while cobalt decreased all enzyme markers at mid-particle to high-particle concentrations . Scanning electron microscopy demonstrated that the morphological response of fibroblasts to titanium included membrane-ruffling and extension of filopodia, typical of active fibroblasts . In contrast, exposure to cobalt at the same concentration resulted in cell crenation, indicative of cell death.

Arch Biochem Biophys, 1993 Jun, 303(2), 355 - 60
Identification and perturbation of mutant human fibroblasts based on measurements of methylmalonic acid and total homocysteine in the culture media; Kolhouse JF et al.; Human and mammalian cells contain two cobalamin-dependent enzymes . Methylmalonyl-CoA mutase isomerizes L-methylmalonyl-CoA to succinyl-CoA in the propionyl-CoA pathway while methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine to form methionine . Decreased activity of mutase leads to an increased methylmalonic acid in the serum of humans while decreased activity of methionine synthase leads to increased homocysteine in the serum of humans . In current studies of cultured fibroblasts, methylmalonic acid levels were specifically increased in media of normal fibroblasts exposed to propionate or in fibroblasts with mutations involving mutase . Homocysteine levels were specifically increased in media of normal fibroblasts exposed to reduced folate concentrations or in fibroblasts involving mutations of methionine synthase . In addition, exposure of normal cells to inhibitory cobalamin analogues resulted in an increase of both methylmalonic acid and homocysteine in the media . This method of analysis appears to be both specific and sensitive for reduced activity of these two enzymes in tissue culture.

Biotechnology (N Y), 1993 Jun, 11(6), 731 - 4
Improved growth and taxol yield in developing calli of Taxus cuspidata by medium composition modification; Fett-Neto AG et al.; Cell culture of Taxus spp . represents a potential alternative source of taxol and related taxanes used in cancer chemotherapy . We have analyzed the effect of different culture media components on growth and production of taxol in developing callus cultures of T . cuspidata . Several sequential modifications were made to the basal B5 medium, which included addition and/or variation in the concentration of sucrose, B5 organic supplements, gibberellic acid, 36 combinations of 2,4-D/kinetin ratios, media salts and organic supplements, phenylalanine, casein hydrolysate and medium pH . The experiments were conducted during a 55 day-growth period followed by taxane extraction and analysis . Significant increases in taxol yield and growth over basal medium grown calli were observed with some of the modified media.

Am J Physiol, 1993 Jun, 264(6 Pt 1), C1594 - 9
Modulation of glucose metabolism in macrophages by products of nitric oxide synthase; Albina JE et al.; Nitric oxide (NO) is a product of L-arginine metabolism that suppresses cellular oxidative metabolism through the inhibition of tricarboxylic acid cycle and electron transport chain enzymes . The impact of NO synthase (NOS) activity on specific pathways of glucose metabolism in freshly harvested and overnight-cultured rat resident peritoneal macrophages, at rest and after stimulation with zymosan, was investigated using radiolabeled glucose . NOS activity was modulated through the L-arginine concentration in culture media and the use of its specific inhibitor, NG-monomethyl-L-arginine, and quantitated using radiolabeled L-arginine . Results demonstrated that NOS activity was associated with increased glucose disappearance, glycolysis, and hexose monophosphate shunt activity and, in line with the known inhibition of oxidative metabolism associated with the production of NO, with a decrease in the flux of glucose and butyrate carbon through the tricarboxylic acid cycle . In addition, the relative increase in glucose utilization that follows zymosan stimulation was enhanced by treatments that suppressed NOS activity . These results demonstrate that the characteristics of glucose metabolism by macrophages are, to a significant extent, determined by products of NOS.

J Biol Chem, 1993 May 25, 268(15), 10881 - 7
Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc; Reinhardt D et al.; Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones . In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin . The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization . Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem . All these fragments were used as soluble and/or immobilized ligands in binding assays . This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan . Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod . Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen . This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain . Most of these short peptides contained several histidines that are likely to mediate binding . Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures . Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures . This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals . Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.

J Biol Chem, 1993 May 25, 268(15), 11208 - 16
Functional erythropoietin receptor of the cells with neural characteristics . Comparison with receptor properties of erythroid cells; Masuda S et al.; Radioiodinated erythropoietin (Epo) was bound specifically to the cells of two non-erythroid clonal lines, PC12 and SN6, which expressed neuronal characteristics . The binding was time-, cell number-, and dose-dependent and was reversible . Although the cloned Epo receptor from PC12 cells (derived from rat adrenal medulla) was identical to that from rat erythroid cells, significant differences in the ligand binding properties between two cell lineages were found; 1) PC12 cells had a single class of binding sites with very low affinity (Kd = 16 nM), whereas erythroid cells had two classes of binding sites with different affinities (Kd = 95 pM for high affinity sites and 1.9 nM for low affinity sites), and 2) cross-linking experiments revealed one cross-linked product of 105 kDa for PC12 cells and two products of 140 and 120 kDa for erythroid cells . Taken together with additional results, the presence of a putative accessory protein(s) that may alter the ligand binding affinity through interaction with Epo receptor is discussed . The binding of Epo to PC12 cells caused a rapid increase in the cytosolic concentration of free calcium . The presence of EGTA had no effect on the Epo binding but completely inhibited the calcium increase, indicating that Epo stimulated the calcium influx from outside of the cells . The addition of Epo to the culture media of PC12 cells elevated the intracellular concentrations of monoamines.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 297 - 301
Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli; Chang YF et al.; Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi . This protein induces immunity against infection in mice . The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E . coli has not yet been reported . In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E . coli when it also harbors the hemolysin secretion genes hlyBD . The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography . This was possible because the fusion protein contains six histidyl residues in its N-terminus.

Biochemistry, 1993 May 11, 32(18), 4931 - 7
Biosynthesis and processing of endogenous parathyroid hormone related peptide (PTHRP) by rat Leydig cell tumor H-500; Rabbani SA et al.; We have examined in vitro the biosynthesis and processing of endogenous PTHRP in cultured rat H-500 Leydig tumor cells . Cells were grown to confluence and pulse labeled with {3H}Ile, 50 microCi/mL, in Ile free culture medium for 2 min to 6 h . In some experiments incubations were carried out in culture medium alone in the presence of 0.3 mM cycloheximide or 20 micrograms/mL unlabeled Ile . Cell extracts and culture media were analyzed by affinity chromatography employing an antibody directed against the bioactive NH2-terminal region, PTHRP(1-34), followed by gel-permeation or reversed-phase high-performance liquid chromatography . Incorporation of {3H}Ile into PTHRP in cell extracts increased over 20 min during pulse labeling and then remained constant throughout the incubation period up to 6 h . In contrast, the release of {3H}PTHRP into culture medium increased progressively over 6 h . Addition of cycloheximide or unlabeled Ile almost completely blocked incorporation of {3H}Ile into newly synthesised PTHRP . Three molecular forms of PTHRP were seen which comigrated with PTHRP(1-36), PTHRP(1-86), and PTHRP(1-141) standards in both chromatographic systems employed . After 20 min these species comprised approximately 63%, 30%, and 7% of newly synthesized PTHRP, respectively . These three molecular forms of PTHRP were observed both intra- and extracellularly, and no further metabolism of these species was seen after release into conditioned medium . Pulse-chase studies demonstrated a rapid decrease of newly synthesized PTHRP forms within cells after 20 min; there was, however, a progressive increase in {3H}PTHRP in conditioned culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicol Appl Pharmacol, 1993 May, 120(1), 20 - 8
The effect of glutathione depletion on methyl mercury-induced microtubule disassembly in cultured embryonal carcinoma cells; Graff RD et al.; Microtubule (MT) assembly and stability are thought to be dependent on intracellular glutathione for the maintenance of critical sulfhydryl groups . Since methyl mercury (MeHg) is a sulfhydryl-binding toxicant, it is possible that alteration of intracellular glutathione status might enhance the toxic effects of MeHg on microtubules . The influence of MeHg on the relationship between intracellular glutathione and the structural integrity of interphase microtubules was assessed in embryonal carcinoma cells by immunofluorescence microscopy, using antibodies to tyrosinated and acetylated alpha-tubulins . Intracellular glutathione concentrations were reduced by treatment with 10 microM buthionine sulfoximine (BSO; an inhibitor of gamma-glutamyl cysteine synthetase) for 18-24 hr . BSO-treated cells displayed little change in the pattern of microtubule staining, despite reduction of glutathione levels to less than 10% of control levels . Similarly, a combination of BSO and the nonspecific glutathione-depleting agent diethylmaleimide (DEM) had little effect on microtubule networks, except at the highest concentrations of DEM where nonspecific cytotoxicity was observed . The susceptibility of microtubules to MeHg-induced disassembly was determined in normal and glutathione-depleted cells incubated with 1.0 to 7.5 microM MeHg for 2 hr . MeHg treatment alone resulted in concentration-dependent disassembly of microtubules; depletion of glutathione with BSO prior to MeHg treatment did not enhance MT damage . Further, BSO-pretreated cells exposed to MeHg still showed substantial recovery of microtubule networks following removal of MeHg from culture media, even when glutathione levels remained less than 5% of control levels . These data indicate that the integrity of interphase microtubules is largely unaffected by reductions in glutathione concentration and that susceptibility of microtubules to MeHg-induced disassembly is not directly dependent on intracellular glutathione content.

Mol Reprod Dev, 1993 May, 35(1), 24 - 8
Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro; Anbari K et al.; Results of recent experiments indicate that the improved development of mouse embryos in medium containing a low NaCl concentration (85 mM) or the inclusion of the organic osmolyte betaine in a medium containing a high NaCl concentration (125 mM) is correlated with the maintenance of intracellular sodium concentrations that more closely approximate those found in freshly isolated embryos (Biggers et al., 1993, Mol Reprod Dev 34:380-390) . We examined the effect of these different culture media on the relative rates of protein synthesis since increased levels of intracellular sodium inhibit protein synthesis; a reduced rate of protein synthesis could therefore account for the differences in development in the different media, since cell division requires protein synthesis . We observe that the ability of these media to support development and to maintain more physiological concentrations of intracellular sodium is correlated with their ability to support increased relative rates of protein synthesis . Reducing the NaCl concentration from 125 mM to 85 mM leads to a greater fraction of the embryos developing from the 2-cell stage to the 8-cell stage after 1 day of culture and a substantially improves extent of development to the morula stage after 2 days of culture . This reduction in NaCl concentration also leads to a 2.4-fold increase in the relative rate of protein synthesis in 4-cell embryos . Moreover, addition of betaine to medium containing 125 mM NaCl increases the relative rate of protein synthesis . This finding provides an explanation, at least in part, for the increase in development to the blastocyst stage exhibited by mouse embryos cultured in these media.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Pathol, 1993 May, 142(5), 1401 - 8
Fibronectin biosynthesis and cell-surface expression by cardiac and non-cardiac endothelial cells; Johnson CM et al.; We examined the biosynthesis and surface expression of fibronectin, an adhesive glycoprotein, in several types of cultured porcine endothelial cells: pulmonary artery, thoracic aorta, coronary artery, aortic valve, and mitral valve . We used immunocytochemical staining to compare the levels of fibronectin present in these same tissues in vivo . Using endogenous radiolabeling, we found that all cell types except aortic valve endothelial cells synthesized and released into the culture media substantial quantities of fibronectin . Using radioiodination of intact cells, we found that, whereas both thoracic aorta and pulmonary artery cells had measurable fibronectin on the surface, aortic valve, mitral valve, and coronary artery cells had little cell-surface fibronectin present . Immunocytochemical staining showed that all endothelial regions except aortic valve had substantial quantities of immunoreactive fibronectin in vivo . These data suggest that the aortic valve endothelium may be distinct from other endothelia . Such differences could be important for the pathogenesis of valvular disease.

J Invest Dermatol, 1993 May, 100(5), 653 - 9
12-O-tetradecanoylphorbol-13-acetate not only modulates proliferation rates, but also alters antigen expression and LAK-cell susceptibility of normal human melanocytes in vitro; Krasagakis K et al.; For serial cultivation of normal human melanocytes media supplemented with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) are largely employed . By using a culture medium that permits cultivation of melanocytes without TPA, the effects of TPA on melanocyte proliferation, phenotype, and susceptibility to lymphokine-activated killer cells were studied . Addition of 50 ng/ml TPA to the medium induced rapid dendrite formation and increased the cell proliferation rate by 16-63% in mitogen-rich media (four of seven cultures, p < 0.01), and by 237% in mitogen-reduced media (p < 0.001) . Furthermore, several phenotypic changes indicating early stages of melanocyte transformation were induced by 50 ng/ml TPA . These included increased expression of melanoma progression-associated antigens such as A.1.43 and A.10.33, upregulation of nerve-growth factor receptor as well as of the melanocyte-activation marker HMB-45 and of histocompatibility class I antigens . In contrast, the expression of the differentiation marker K.1.2 and of intercellular adhesion molecule-1 was decreased in TPA-treated cultures . Most of these changes persisted even after removal of TPA from the culture medium (> or = 2 weeks) . Staurosporine, a protein kinase C inhibitor, modulated melanocyte-antigen expression similar to TPA, suggesting that protein kinase C downmodulation rather than activation by TPA is involved . In addition to the antigenic alterations, the susceptibility of TPA-treated melanocytes to lymphokine-activated killer cell cytotoxicity decreased by 40% (p < 0.01), possibly due to their altered surface antigen expression . The presented data reveal that the tumor promoter TPA hitherto used as a supplement of melanocyte culture media induces profound phenotypic and functional changes of the cultured cells, indicating incipient transformation of normal human melanocytes in vitro.

Biochem J, 1993 May 1, 291 ( Pt 3), 869 - 73
Sensitive, hydrosoluble, macromolecular fluorogenic substrates for human immunodeficiency virus 1 proteinase; Anjuere F et al.; Hydrosoluble macromolecular fluorogenic substrates specific for the human immunodeficiency virus 1 (HIV-1) proteinase have been prepared . The fluoresceinyl peptide Ftc-epsilon-Ahx-Ser-Phe-Asn-Phe-Pro-Gln-Ile-Thr-(Gly)n, corresponding to the first cleavage site of HIV-1 gag-pol native precursor was linked to a water-soluble neutral (Lys)n derivative . The epsilon-aminohexanoyl residue (epsilon-Ahx) and the glycyl sequence were added in order to improve the stability of the substrate and the accessibility of the cleavage site to the HIV-1 proteinase respectively . This macro-molecular peptidic-substrate conjugate is significantly more water-soluble than the free peptide itself on a substrate molar concentration basis . The assay is based on the quantitative precipitation of the polymeric material by adding propan-2-ol whereas the fluorescent peptide moiety released upon proteolysis remains soluble in the supernatant . The proteinase activity is assessed by measuring the fluorescence of the supernatant . This assay allows the detection of a few fmol of HIV-1 proteinase, even in the presence of cell culture media, plasma or cell lysate and it gives accurate results within a large proteinase concentration range . The hydrosoluble macromolecular substrate is also suitable for determining the HIV-1 proteinase activity using 96-well microplates, allowing us to test accurately and rapidly numerous enzyme samples and/or the potency of new proteinase inhibitors.

J Cell Physiol, 1993 May, 155(2), 323 - 32
Mechanism of retinoid-induced activation of latent transforming growth factor-beta in bovine endothelial cells; Kojima S et al.; Cell-associated plasmin is a putative physiological activator of latent transforming growth factor-beta (LTGF-beta) . Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell-associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF-beta using bovine endothelial cells (ECs) as a model system . ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF-beta in the culture media in a dose- and time-dependent fashion . Cells were treated with 2 microM retinol for 24 h, and the amount of TGF-beta produced during a subsequent 12-h incubation period was measured . Out of a total of 14 pM LTGF-beta secreted, 0.7 pM was converted to active TGF-beta . Northern blot analyses showed that mRNA levels for TGF-beta 2 but not for TGF-beta 1 increased in cells treated with retinol . Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF-beta, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF-beta in retinoid-stimulated cells . Antibody against the LTGF-beta binding protein blocked activation implying that localization of LTGF-beta through its binding protein may be important . However, inhibition of binding of LTGF-beta to the cell surface mannose 6-phosphate receptor did not prevent activation . These data indicate that retinoids up-regulate the production of LTGF-beta in ECs and induce activation of LTGF-beta, perhaps, by increasing PA and plasmin levels . Thus, TGF-beta might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro.

J Gastroenterol Hepatol, 1993 May-Jun, 8(3), 228 - 31
Enhanced production of leukotriene B4 by peripheral blood mononuclear cells in patients with fulminant hepatitis; Asano F et al.; The production rate of leukotriene B4 (LTB4) was measured using peripheral blood mononuclear cells (PBMC) in patients with fulminant hepatitis (FH) or other liver diseases . LTB4 in the culture media of PBMC under stimulation with Ca-ionophore was fractionated by HPLC and measured by radioimmunoassay . The production rate of LTB4 was elevated in 16 of 17 FH patients (3.3 +/- 0.2 ng/10(6) cells for 5 min), while the production was below detectable level in patients with acute or chronic hepatitis and in healthy controls . In FH patients, the highest production rate of LTB4 was observed in the initial period of the disease . Enhanced LTB4 production may indicate the primed state of PBMC--the primed mononuclear cells are regarded as participating in the development of massive liver necrosis and of other organ failures in FH.

Fertil Steril, 1993 May, 59(5), 1022 - 7
Hysterosalpingography contrast media and chromotubation dye inhibit peritoneal lymphocyte and macrophage function in vitro: a potential mechanism for fertility enhancement; Goodman SB et al.; OBJECTIVE: To determine the effects of hysterosalpingography (HSG) contrast media (CM) and chromotubation dye on peritoneal lymphocyte proliferation and macrophage phagocytosis in vitro . DESIGN: Peritoneal fluid (PF) lymphocytes and macrophages were isolated from 40 subfertile women undergoing diagnostic laparoscopy and 12 fertile women having laparoscopic tubal ligation . Dilutions of renografin, ethiodol, methylene blue, and indigo carmine were added to peritoneal lymphocyte and macrophage cultures . Tissue culture media alone served as control . Lymphocyte proliferation was assessed by hemocytometer counts and 3H-thymidine incorporation . Macrophage function was determined by phagocytosis of fluorescent microspheres . RESULTS: Peritoneal lymphocyte proliferation and macrophage phagocytosis were significantly inhibited by renografin, ethiodol, methylene blue, and indigo carmine in a dose-dependent manner . CONCLUSION: Inhibition of PF immune cell function by HSG CM and chromotubation dye may provide a potential mechanism for fertility enhancement after these diagnostic procedures.

J Biochem (Tokyo), 1993 May, 113(5), 531 - 7
Plasma kininogen deficiency: associated defective secretion of kininogens by primary cultures of hepatocytes from brown Norway Katholiek rats; Hayashi I et al.; To clarify the mechanism of plasma kininogen deficiency of Brown Norway Katholiek strain (B/N-Katholiek) rats, we compared synthesis and secretion of kininogens by primary cultures of hepatocytes from B/N-Katholiek and B/N-Kitasato (normal strain) rats . Pulse-and-chase experiments using {35S}methionine demonstrated that kininogen antigens with molecular masses of 100 and 66 kDa, corresponding to high- and low-molecular-weight kininogens (HK and LK), respectively, were detected in the hepatocytes of both strains . These proteins were then processed to 108- and 71-kDa forms, respectively, and secreted by the normal hepatocytes, while the latter forms were hardly secreted in the culture media of the deficient hepatocytes . However in the deficient cells, 100- and 66-kDa forms were accumulated, but 108- and 71-kDa bands were faint . A subcellular fractionation study showed that a relatively higher amount of the kininogen antigens was present in the lysosomal fraction of B/N-Katholiek hepatocytes than in that of B/N-Kitasato hepatocytes . From these results we postulate the cause of the secretion defect of B/N-Katholiek liver to be as follows . (i) B/N-Katholiek liver could synthesize the mature secretable forms of HK and LK, but they are too rapidly transported to the lysosomes, or (ii) the mature forms in B/N-Katholiek hepatocytes might be synthesized much more slowly than those in the normal cells . T-Kininogen was normally synthesized and secreted by the hepatocytes of B/N-Katholiek, suggesting that the secretion defect could be limited to HK and LK, at a common site.

Clin Exp Allergy, 1993 May, 23(5), 377 - 83
Allergenicity of the cat flea (Ctenocephalides felis felis)
Trudeau WL, Fernandez-Caldas E, Fox RW, Brenner R, Bucholtz GA, Lockey RF.
Adult fleas, spent and unspent culture media were extracted and the radio-allergosorbent test (RAST) performed with sera of 48 cat flea skin test-positive individuals from the Tampa Bay area of Florida . Sixteen sera (33.6%) had a positive RAST to the cat flea extract prepared in our laboratory {1.7-11.4% of the total counts (TC) added} . Six of the 16 sera (12.5%) also contained specific IgE to allergens in the spent medium (0.8-3.3% TC) . The allergen composition and strength were studied by RAST inhibition of two commercial cat flea extracts and compared with our in-house flea extract . The results demonstrated similar allergen compositions and different potencies . Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the in-house flea extract showed several Coomassie blue-stained bands (10-85 kD) . SDS-PAGE immunoblots revealed five IgE-binding bands at 34, 35, 39, 54 and 60 kD . Flea allergens were quantified in eight house dust samples using RAST inhibition assays and expressed as RAST inhibition units; five of these samples contained detectable levels . Cat flea allergens may contribute to the allergenicity of house dust in areas of heavy flea infestation.

Exp Lung Res, 1993 May-Jun, 19(3), 315 - 25
Fluorescence anisotropy as an index of fetal lung maturation in vitro; Gewolb IH et al.; Fluorescence techniques have been used to assess the viscosity of surfactant-containing fluids in vivo and have been successfully employed clinically as indices of lung maturity . However, fluorescence measurements have not been previously used as indicators of fetal lung maturation in an in vitro system . Lung explants derived from 19-, 20-, and 21-day fetal rats were cultured in F-12 medium for 24-72 h . Tissue homogenates and culture medium were eluted on Sephacryl S-300 columns, a diphenylhexatriene (DPH) probe was added to each fraction, and fluorescence anisotropy and intensity were measured after excitation at 357 nm and emission at 435 nm . Elution fractions containing the major fluorescence peak were demonstrated to correspond to the phosphatidylcholine-containing fraction and were shown to contain lamellar bodies . Fluorescence anisotropy of tissue homogenates obtained from 19-day lung explants decreased after 72 h in culture, suggesting lower microviscosity of the surfactant-containing fractions . Assessment of culture media collected at 24-h intervals revealed significant decreases in anisotropy by 48 h for the 19-day explants, and by 24 h for the 20- and 21-day explants . Anisotropy of the final (48-72 h) culture media aliquots was significantly lower for 21-day explants (0.144 +/- 0.004, SE), than for 20-day (0.172 +/- 0.013) or 19-day explants (0.197 +/- 0.008), p < .005 . Anisotropy of culture medium tended to be lower than anisotropy of corresponding tissue homogenates, suggesting that viscosity of recently secreted surfactant may be different from viscosity of surfactant within lamellar bodies in type II cells . Relative fluorescence intensity of tissue homogenates also increased with time in culture . These results indicate that fluorescence anisotropy can be used to assess the viscosity of surfactant in vitro and serve as another index of fetal lung maturation in in vitro systems . Estimation of the microviscosity of the surfactant phospholipid bilayer using anisotropy measurements may provide additional insight into such roles of surfactant function as adsorption and spreading.

Hum Reprod, 1993 May, 8(5), 678 - 83
In-vitro synthesis of total protein and placental protein PP14 by the fallopian tube mucosa: variation in relation to anatomical site, the ovarian cycle and the menopause; Maguiness SD et al.; De-novo synthesis and secretion of protein by short term explants of mucosa from each anatomical section of the Fallopian tube and endometrium of pre-menopausal (n = 25) and tubal mucosa of post-menopausal (n = 5) women were studied by demonstration of incorporation of radiolabelled L-{35S}methionine and one-dimensional SDS-polyacrylamide gel electrophoresis . A consistent finding in 25 pre-menopausal women was the presence of a 25 kDa protein band synthesized by tissue obtained throughout the ovarian cycle . Western blotting demonstrated that this protein band contained placental protein 14 (PP14)-like immunoreactivity in the proliferative (n = 2) and luteal phase (n = 2) of the ovarian cycle . To determine if there is quantitative variation in total protein and PP14 synthesis and secretion during the ovarian cycle, the total quantities of protein and PP14 synthesized were determined by Coomassie Brilliant Blue staining and radioimmunoassay respectively . Analysis of the results of total protein assay revealed statistically significant differences in relation to the anatomical origin of the study tissue (P < 0.01), the stage of the ovarian cycle (P < 0.04) and the manner in which each anatomical site varied during the ovarian cycle (P < 0.01), the endometrium being significantly different from the Fallopian tube . When the data for PP14 synthesized by the Fallopian tube mucosa were analysed, these effects were not seen . PP14 was not detected in the culture media of Fallopian tube mucosa obtained from post-menopausal women.

Boll Soc Ital Biol Sper, 1993 May, 69(5), 281 - 5
{The concentration of osteocalcin in the culture media of bone cultured in vitro subjected to intermittent mechanical load with the addition of 1,25(OH)2D3}; Lozupone E et al.; The osteocalcin concentration decreases in the medium of bones submitted to intermittent mechanical forces in organotypic culture (6) . The functional significance of this behaviour is unknown . To investigate the dynamics of the osteocalcin production, we added 1,25(OH)2D3 to the culture medium to stimulate osteoblastic production of the osteocalcin . The bones were isolated from 12 day-old rats, placed in culture medium added with 10(-8) M 1,25(OH)2D3 and subjected 1/2 h daily to an intermittent mechanical load (11.6 Kg/mm2) at 1 Hz frequency . 12, 24 and 48 hours after the loading the alcaline phosphatase activity (p-nitrophenol as substrate) and the osteocalcin concentration (RIA method) were evaluated in the media . The phosphatase activity does not change in the control bones, while it significantly increases in the loaded bones 24 h after loading (fig . 1) . The osteocalcin concentration increases significantly in the control bones, with a peak at 24 h; in the loaded ones, it does not increase at 24 h with respect to 12 h, while it increases 48 h after loading, but at a lower level than the control bones . Since the increase of the alcaline phosphatase activity is a sign of new bone production, our results indicate that an osteoid deposition is likely to take place in the loaded bones, not in the control ones . The increase in the control bones of osteocalcin concentration is in agreement with the results of other researches (7), and indicates that the osteoblasts of this experimental system are able to produce osteocalcin under appropriate conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biol Signals, 1993 May-Jun, 2(3), 155 - 65
Characterization of cell types within a chorionic gonadotropin-secreting, mechanically dissociated human placental cell population in perifusion; Steele GL et al.; A cell perifusion system was established to examine human placental endocrine regulation by locally synthesized peptides . First-trimester and term trophoblast cells were mechanically dissociated . Cells were plated on microcarrier beads and cultured for 7-14 days . Cells on beads were loaded in chambers, perifused with culture media and effluent was assayed for chorionic gonadotropin (hCG) . Mechanical dissociation of placental tissue produced cell preparations with 85-95% viability . Staining with Masson trichrome, cytokeratin and beta-hCG antibodies suggested that greater than 50% of the cells were trophoblast . Perifused trophoblast cells secreted hCG in a continuous non-pulsatile fashion, independent of exogenous hormonal stimuli . hCG secretion from first-trimester trophoblast cells remained stable in static culture for 14 days . GnRH perifusion (10(-8) M) for 15-120 s transiently increased hCG secretion from first-trimester trophoblast cells . Longer GnRH exposure stimulated greater hCG secretion . Each of 3 consecutive pulses of 8-bromo-cyclic adenosine-3',5'-monophosphate (cAMP, 10(-9) M) administered at 2-hour intervals stimulated transient hCG secretion from first-trimester and term placental cells . cAMP stimulated hCG secretion more potently from first-trimester than from term placental cells.

Biol Trace Elem Res, 1993 May-Jun, 37(2-3), 187 - 99
Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress; Richard MJ et al.; In purified system zinc has been shown to have an antioxidant role . Its effects on the resistance of cultured cells towards oxidative stress in vitro were examined . Diploid human skin fibroblasts were grown for 21 d in culture media (RPMI 1640 containing 15% fetal calf serum) added with different zinc (Zn) concentrations (100, 125, and 150 microM as Zinc chlorur ZnCl2) . In comparison, cell controls were grown in standard culture media (6.5 microM Zn) . The intracellular zinc levels of treated fibroblasts increased from 3- to 7-fold (2330 +/- 120 ng/mg protein in 150-microM Zn-treated cells versus 331 +/- 21 ng/mg protein in control cells) . The intracellular copper increased 3- fold whereas the iron content slightly but not significantly decreased . The index of basal lipid peroxidation measured as thiobarbituric acid reactants (TBARs) of zinc-supplemented cells was lower than that of non zinc supplemented controls (0.89 mumol/g protein in 150 microM Zn-treated cells versus 1.59 mumol/g protein in controls) . At these high doses of zinc, fibroblasts expressed lower antioxidant metalloenzymes activities . Diminished TBARs in Zn treated cells tends to support that Zn acts protectively against free radical mediated damage . However when the cells were challenged with extracellular oxidant stresses mediated by hypoxanthine/xanthine oxidase or hydrogen peroxide (H2O2), an increased toxicity in Zn-supplemented cells was observed . When we applied an intracellular oxidative stress as UV-B or UV-A radiation, Zn-treated fibroblasts were more resistant than cells grown in normal medium . If Zn has shown antioxidant effect in some in vitro or in vivo systems our observations clearly demonstrate that this role is not mediated by antioxidant metalloenzymes.

In Vitro Cell Dev Biol Anim, 1993 May, 29A(5), 403 - 7
Vitronectin secretion by hepatic and non-hepatic human cancer cells; Yasumitsu H et al.; Thirty-eight human cancer cell lines and subclones derived from 12 different organs were screened for vitronectin secretion in their culture media . By immunoblotting analysis we detected high secretion by three out of five hepatoma cell lines tested but no secretion by the others . In addition, significant secretion was observed in seven non-hepatic cancer cell lines and subclones derived from the cervix, lung, and pancreas . These vitronectin-secreting cells included PLC/PRF/5, HuH-6 #5, HuH-7, HeLa S3, HeLa.P3 #2, #3, #6, #8, A549, and MIAPaCa-2 . The results were further confirmed by quantitative analysis using sandwich enzyme-linked immunoassay, and activity analysis of cell attachment promotion on Western blotted filters.

Am J Physiol, 1993 May, 264(5 Pt 1), C1345 - 9
A dramatic pH-dependent alteration in ANP receptor density: a note for using cultured cells; Katafuchi T et al.; Culture media tend to become acidic when rapidly growing cells are cultured under batch conditions using a CO2/HCO3- buffer system . The effects of this inherent lowering of pH on cellular makeup of cultured cells, which have long been ignored, were examined by monitoring the pH and number of the atrial natriuretic peptide (ANP) receptors expressed on the cultured bovine endothelial cells . The Eagle's minimum essential medium was adjusted to three different pH values of 7.0, 7.4, and 7.7 and used for 48-h batch cultures . After this 48-h incubation, the pH values of the media were found to be 7.0, 7.1, and 7.4, respectively . These pH shifts had unexpectedly strong influences on the ANP receptor levels without affecting the affinity . Cells maintained in the slightly higher pH medium had a trace amount of the receptor (< 10 sites/cell), while those in the lower pH environment exhibited a large number of binding sites (40,000 sites/cell) . Similar situations might occur in other cellular components and in other types of cells, and therefore, such possibilities should be kept in mind when cultured cell systems are used.

Eur J Pharmacol, 1993 Apr 15, 245(2), 147 - 56
Molecular cloning and characterization of the angiotensin receptor subtype in porcine aortic smooth muscle; Itazaki K et al.; A cDNA encoding porcine aortic smooth muscle angiotensin II (AII) receptor has been isolated using the homology screening approach and sequenced . Specific binding of {125I}AII was found in COS-7 cells transfected with the cDNA (Kd = 0.37 nM, Bmax = 1 approximately 3 x 10(4)/cell) and was displaced with unlabeled AII-related peptides and DUP753 in the order of {Sar1,Ile8}AII = AII > des-Asp1-{Ile8}AII = DUP753 > angiotensin I = angiotensin III . EXP655 had no effect on {125I}AII binding . COS-7 cells transfected with the cDNA responded to AII by only a small increase in the concentration of intracellular free Ca2+ . However, electrophysiological study of the receptor expressed in Xenopus laevis oocytes provided strong evidence that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of Ca2+ . Northern blot analysis in cultured porcine aortic smooth muscle cells demonstrated that the expression of this gene varies with the culture media . These results indicate that the cDNA encodes the functional and regulated AT1 subtype of AII receptors.

J Biol Chem, 1993 Apr 15, 268(11), 7650 - 9
Utilization of exogenously supplied sphingosine analogues for sphingolipid biosynthesis in Chinese hamster ovary and mouse LM cell fibroblasts; Ladenson RC et al.; Growth of Chinese hamster ovary and LM cells is inhibited by relatively low concentrations of sphingosine in the culture media . This effect is diminished by an order of magnitude by conversion of this positively charged long-chain amino alcohol to a number of N-acetylated analogues, such as N-acetylsphingosine, N-acetylsphingosine phosphate, and N-acetylsphingosine phosphorylcholine . Synthesis of sphinganine and its incorporation into ceramide, sphingomyelin, and glycosphingolipids (GSL) was monitored using a short pulse of {14C}serine together with a long pulse of {3H}-galactose . Compared to unsupplemented cultures, growth with 15-30 microM N-acetylsphingosine suppressed incorporation of 14C radioactivity into ceramide, sphingomyelin, and GSL by 75-95% without accumulation of labeled sphinganine and without any appreciable change in membrane phospholipid or total GSL content . Furthermore, when cells were cultivated with 15 microM {4,5-3H}N-acetylsphinganine to monitor its utilization for sphingolipid synthesis, considerable loss of radiolabel occurred due to desaturation of sphinganine to sphingosine . Nevertheless, most of the residual label was found in the long-chain base and not the acyl group of sphingomyelin, indicating that the exogenously supplied base was utilized intact for complex lipid synthesis . Radiolabel was also found in ceramide and glycosphingolipid fractions . Thus, established cell lines whose growth is very sensitive to long-chain amino alcohols can be cultivated with sphinganine (sphingosine) analogues at concentrations which suppress endogenous sphinganine production but support continued synthesis of complex sphingolipids.

J Neurosci Res, 1993 Apr 15, 34(6), 622 - 8
Characterization of a transmissible growth-promoting agent derived from CSF of schizophrenic patients which is active on human neuroblastoma cells; Shirabe S et al.; A growth-promoting agent for the human neuroblastoma cell line SK-N-SH(EP) (SH-EP) has been detected in human cerebrospinal fluid (CSF) derived from schizophrenic patients . Following treatment with the CSF, a number of properties of the SH-EP cells changed permanently . These included an accelerated rate of growth, an increased cell density at confluence, a change of cell shape, and an increased ability to form colonies in soft agar . All of these changes are consistent with further cellular transformation of the SH-EP cells . Once the cells' properties had changed following CSF treatment, the growth-promoting activity was found to be present in freeze-thawed cell extracts and in the culture medium, and could be passed to untreated SH-EP cells . The activity could be detected in culture media diluted as high as 10(8) . It was inactivated by proteinases, chloroform, or heat but passed through a 0.22-micron filter . The growth-promoting activity can be banded on a Percoll gradient, suggesting that it is particulate rather than a soluble growth factor.

Rinsho Ketsueki, 1993 Apr, 34(4), 423 - 6
{Growth characteristics of a human myeloma cell line transfected with IL-6 cDNA}; Takahashi T et al.; It remains to be clarified whether IL-6 acts on the growth of human myeloma cells by an autocrine or paracrine mechanism . Even in established myeloma cell lines, the autocrine growth by IL-6 appears unusual . In the present study, we deviced a model of IL-6 autocrine growth in vitro by transfecting IL-6 cDNA into a human myeloma cell line that had a proliferative response to IL-6 but did not produce IL-6 . After IL-6 transfection, the cells (S6B45) proliferated in culture media without IL-6 . IL-6 production by S6B45 was demonstrated both at protein and mRNA level . The growth of S6B45 was definitely inhibited by anti-IL-6 (MH166) or anti-IL-6 receptor (PM1) monoclonal antibodies . Furthermore, S6B45 was successfully transplanted to nude mice . The transplanted tumor growth was clearly inhibited by the administration of MH166 or PM1 to the mice . The in vivo antitumor activity of these antibodies suggest a new therapeutic strategy against tumors that proliferate by an autocrine mechanism through a cytokine such as IL-6.

Thromb Haemost, 1993 Apr 1, 69(4), 387 - 91
Role of urokinase type plasminogen activator (u-PA) in corneal epithelial migration; Morimoto K et al.; The role of plasminogen activator (PA) in the migration of corneal reepithelialization was studied . Rabbit corneal blocks were cultured, and both the extent of epithelial migration over the exposed corneal stroma and the activity of PA released into the culture media were measured . A significant, direct correlation between epithelial migration and PA activity in the medium was observed, even when the migration was stimulated by fibronectin or EGF, or was inhibited by cytochalasin B or cycloheximide . Zymography confirmed that the PA released into the culture medium was of the urokinase type (u-PA) . Immunohistochemical studies showed that u-PA and plasmin(ogen) were present at the leading edge of the migrating epithelium . Studies of corneal cell cultures indicated that epithelial cells rather than endothelial cells or fibroblasts were the source of the u-PA . The addition of antihuman u-PA IgG or protease inhibitors retarded the migration of the corneal epithelium in a dose-dependent manner, indicating that u-PA activity is essential for the migration of the corneal epithelium . These findings suggest that the migration of corneal epithelial cells requires not only cell attachment to the extracellular matrix through the fibronectin but also degradation of the fibronectin by the release of cellular u-PA.

J Wildl Dis, 1993 Apr, 29(2), 214 - 8
Experimental Borrelia burgdorferi infections in the white-footed mouse, deer mouse, and fulvous harvest mouse detected by needle aspiration of spirochetes; NieLin G et al.; Three methods were tested for recovering Borrelia burgdorferi from live mice onto BSK II culture medium . Four laboratory-reared Peromyscus leucopus were inoculated intraperitoneally with the JD-1 isolate of Borrelia burgdorferi . Borrelia burgdorferi spirochetes were recovered from 13 of 20 (65%) samples taken by needle aspiration between days 7 and 40 post-inoculation (PI) and from 1 of 16 samples of skin obtained by ear punch biopsy during the same sampling period . Spirochetes were not recovered from culture media inoculated with mouse blood . The use of needle aspirates for recovering spirochetes was compared among three species of mice: P . leucopus, P . maniculatus, and Reithrodontomys fulvescens . Spirochetes were isolated from 14 of 15 aspiration samples from four P . maniculatus, 12 of 20 from three P . leucopus, and 15 of 20 from four R . fulvescens taken between days 7 and 48 PI . Spirochetes were isolated from only one aspiration sample between days 80 and 95 PI from any of the mice tested . Needle aspiration was an efficient method for repeated recovery of B . burgdorferi from live, experimentally infected mice . We also document R . fulvescens as an experimental host for B . burgdorferi . Based on their susceptibility to infection, all species of mice tested herein may play a role in the epidemiology of Lyme disease where their distribution is compatible with endemic transmission.

Am J Physiol, 1993 Apr, 264(4 Pt 1), G601 - 8
Prostaglandin E2 downregulates Kupffer cell production of IL-1 and IL-6 during hepatic regeneration; Goss JA et al.; The mammalian liver possesses the ability to regenerate to its original size after a 70% partial hepatectomy (PHx) . The capacity of rat Kupffer cells (KC) isolated at specific intervals after PHx to produce interleukin (IL)-1, IL-6, and prostaglandin E2 (PGE2) in response to endotoxin {lipopolysaccharide (LPS)} stimulation was evaluated in standard RPMI 1640 (1,200 microM L-arginine) and arginine-depleted RPMI 1640 (< 10 microM L-arginine) media . Because KC function in an environment in which high arginase activity results in negligible L-arginine levels, the 10 microM L-arginine RPMI 1640 was used to simulate the hepatic microenvironment . Regenerating liver KC 12-120 h after PHx responded to LPS with a significantly greater (P < 0.05) production of IL-1 and IL-6 in standard RPMI 1640 . This enhancement of regenerating liver KC to produce IL-1 and IL-6 was increased (P < 0.05) by placing these same KC in 10 microM arginine RPMI 1640 culture media . During the same time period, regenerating liver KC produced significantly elevated (P < 0.01) PGE2, again with greater differences in the low-arginine media . In vivo KC PGE2 blockade by indomethacin (5 mg/kg) significantly (P < 0.05) inhibited hepatic regeneration . When the cyclooxygenase inhibitor indomethacin (10 microM) was added to cultures, the production of PGE2 by KC was prevented, and in arginine-depleted cultures, IL-1 and IL-6 production was upregulated (P < 0.05) . We conclude that during hepatic regeneration, KC IL-1 and IL-6 production is elevated and is controlled in an autoregulatory fashion by elevated KC PGE2 production.

Am J Pathol, 1993 Apr, 142(4), 1265 - 78
Effects of interferon-gamma on primary cultures of human brain microvessel endothelial cells; Huynh HK et al.; Primary cultures of human brain microvessel endothelial cells were used to study the effects of human recombinant interferon-gamma (IFN-gamma) on cerebral endothelium in vitro . Incubation of monolayers with various concentrations of IFN-gamma (10 to 200 U/ml) for 12 to 96 hours induced surface expression of class II major histocompatibility complex (Ia) antigen in a time- and concentration-dependent manner . In immunogold-stained cultures, labeling was observed as early as 12 hours, was maximal after 48 hours, and persisted at plateau levels in the continuous presence of the cytokine . Expression was blocked by coincubation with anti-IFN-gamma antibody and was reversed 4 days following removal of IFN-gamma from the culture media . Endothelial cells treated with IFN-gamma for 3 to 4 days became spindle-shaped, extensively overlapped, and frequently formed cellular whorls . These changes did not occur in the presence of anti-IFN-gamma antibody and reversed upon removal of IFN-gamma from the media . The morphological alterations were associated with increased permeability of confluent monolayers to macromolecules as compared with untreated cultures . The results of these studies indicate that human brain microvessel endothelial cells respond to in vitro cytokine stimulation by undergoing profound morphological, functional, and permeability changes . We conclude that cerebral endothelium may play an important role in the initiation and regulation of lymphocyte traffic across the blood-brain barrier in inflammatory disorders of the human central nervous system.

FASEB J, 1993 Apr 1, 7(6), 586 - 91
Proline-mediated enhancement of hepatocyte function in a collagen gel sandwich culture configuration; Lee J et al.; Isolated rat primary hepatocytes were cultured between two layers of gelled collagen in a sandwich configuration that reinstates the cellular polarity necessary for long-term function in vitro . Maintenance of hepatocyte function, as measured by the secretion of albumin, was shown to be dependent on both the sandwich gel configuration and the continued presence of L-proline in the culture media . Cis-hydroxyproline, an analog of proline known to prevent the proper folding of triple helical collagen molecules, inhibited the response of sandwiched hepatocytes to proline in a dose-dependent and reversible manner . The addition of cis-hydroxyproline to cultures established for 7 days also resulted in the inhibition of hepatocyte function . These data support the hypothesis that continued collagen synthesis by hepatocytes is critical for hepatocyte function in the sandwich gel configuration.

Mol Reprod Dev, 1993 Apr, 34(4), 380 - 90
The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro; Biggers JD et al.; The development of outbred mouse (CF1) zygotes in vitro has been studied using medium SOM in which the concentrations of NaCl (85, 105, 125 mM), glutamine (0, 1, 2 mM), and betaine (0, 1, 2 mM) were varied . The effects of the compounds were studied using a 3(3) factorial experimental arrangement . The inhibitory effect of relatively high concentrations of NaCl and the protective effect of glutamine were confirmed . Betaine, an organic osmolyte, can also protect against the deleterious effects of relatively high concentrations of NaCl . The intracellular contents of potassium and sodium have also been measured in single zygotes using X-ray electron probe spectrometry . When medium SOM contains 85 mM or 125 mM NaCl, the intracellular content of Na rises and the content of K decreases . These changes are partially reduced in the presence of 125 mM NaCl if betaine is also in the medium . Betaine has no effect on the intracellular content of K and Na if the concentration of NaCl is 85 mM . These results suggest that organic osmolytes may be required in embryo culture media to prevent excessive changes in the intracellular ionic concentration.

Free Radic Biol Med, 1993 Apr, 14(4), 371 - 9
Cytostatic effects of horseradish and thyroid peroxidase derived free radicals; Moore KL et al.; An otherwise noncytostatic flux of H2O2 from glucose and glucose oxidase became cytostatic to cultured Chinese Hamster Ovary (CHO) cells when horseradish or thyroid peroxidase was added to the culture medium . Electron spin resonance (ESR) measurements showed that one or more factors present in the culture medium promote the one-electron oxidation of a reduced nitroxide or glutathione in an H2O2/peroxidase-dependent process . Moreover, a reduced nitroxide conferred significant protection against the cytostatic effect of H2O2/peroxidase . Cytostatic effects were not only seen in the presence of the active H2O2/peroxidase system, but also in media which had been preexposed to H2O2/peroxidase but no longer contained an active H2O2 generating system . It is suggested that peroxidases oxidize one or more factors in tissue culture media to free radicals, which react with nearby components of cells or form toxic products, causing growth inhibition . If similar free radical precursors are present in tissue fluids, some of the toxicity of H2O2 in vivo may be due to peroxidase-mediated endogenous free radical generation.

J Parasitol, 1993 Apr, 79(2), 160 - 6
Synthesis of tyrosine-derived cross-links in Ascaris suum cuticular proteins; Fetterer RH et al.; Tritiated dityrosine and isotrityrosine were detected by high performance liquid chromatography (HPLC) of acid hydrolysates of cuticular proteins from larval Ascaris suum following their 96-hr in vitro incubation in {3H}tyrosine . Sixty percent of the HPLC-recovered radiolabel was present as tyrosine, 20% as dityrosine, and 6% as isotrityrosine . Approximately 13% of radioactivity was associated with several unidentified peaks . A similar distribution of radioactivity was observed in acid hydrolysates of cuticular proteins from young adults of A . suum following 48 hr in vitro incubation with {3H}tyrosine . The 2-mercaptoethanol (2ME)-insoluble cuticular protein from the larval stages had a higher rate of synthesis of {3H}dityrosine than did the 2ME-soluble cuticular proteins, whereas the 2ME-soluble cuticular proteins had higher rates of synthesis of {3H}isotrityrosine . Pulse-chase studies of A . suum larvae demonstrated a relatively low rate of synthesis of both dityrosine and isotrityrosine . The addition to the culture media of the peroxidase inhibitors, phenylhydrazine (PHEN), 3-amino-1,2,4-triazole (AT), and N-acetyltyrosine (NAT) reduced the amount of {3H}tyrosine synthesized into both dityrosine and isotrityrosine . In a cell-free system, soluble extracts of A . suum larvae also converted radiolabeled tyrosine to dityrosine; isotrityrosine was produced by some extracts . The rate of conversion correlated with time of incubation and the volume of added extract and was inhibited by AT, NAT, and PHEN, with PHEN being the most potent inhibitor . The results of the present study suggest that the tyrosine residues of the cuticular proteins are posttranslationally modified by the formation of dityrosine and isotrityrosine cross-links . This modification is most likely mediated by a peroxidase.

J Neurochem, 1993 Apr, 60(4), 1578 - 81
Chronic ethanol exposure potentiates NMDA excitotoxicity in cerebral cortical neurons; Chandler LJ et al.; The effect of acute and chronic ethanol exposure on excitotoxicity in cultured rat cerebral cortical neurons was examined . Neuronal death was quantitated by measuring the accumulation of lactate dehydrogenase (LDH) in the culture media 20 h after exposure to NMDA . Addition of NMDA (25-100 microM) to the culture dishes for 25 min in Mg(2+)-free buffer resulted in a dose-dependent increase in LDH accumulation . Phase-contrast microscopy revealed obvious signs of cellular injury as evidenced by granulation and disintegration of cell bodies and neuritic processes . Chronic exposure of neuronal cultures to ethanol (100 mM) for 96 h followed by its removal before NMDA exposure, significantly increased NMDA-stimulated LDH release by 36 and 22% in response to 25 microM and 50 microM NMDA, respectively . Neither basal LDH release nor that in response to maximal NMDA (100 microM) stimulation was altered by chronic alcohol exposure . In contrast to the effects of chronic ethanol on NMDA neurotoxicity, inclusion of ethanol (100 mM) only during the NMDA exposure period significantly reduced LDH release by approximately 50% in both control and chronically treated dishes . This reduction by acute ethanol was also observed under phase-contrast microscopy as a lack of development of granulation and a sparing of disintegration of neuritic processes . These results indicate that chronic exposure of ethanol to cerebral cortical neurons in culture can sensitize neurons to excitotoxic NMDA receptor activation.

Rev Argent Microbiol, 1993 Apr-Jun, 25(2), 59 - 69
{Effect of water activity on the growth of the xerophilic mold Eurotium herbariorum}; Vaamonde G et al.; The influence of water activity (aw) on growth of a xerophilic mold isolated from dried figs and identified as Eurotium herbariorum was studied on culture media of aw adjusted with sucrose or glycerol . Rate of radial growth (kr) and lag period were the kinetic parameters analyzed . Fungal growth was inhibited at aw > 0.97 . In the presence of sucrose, optimum growth was found in M6OY agar (malt agar with yeast extract and 60% W/V of sucrose, aw = 0.95) . On glycerol (aw ranging from 0.65 to 0.90) the fungus did not grow at aw < 0.80 . Sucrose supported better growth than glycerol at aw 0.90.

J Anim Sci, 1993 Apr, 71(4), 923 - 9
Validation of a culture system for porcine pituitary cells: effects of growth hormone-releasing factor and(or) somatostatin on growth hormone secretion; Farmer C et al.; The goal of the study was to establish the age-related responses of cultured porcine pituitary cells to growth hormone-releasing factor (GRF) and(or) somatostatin (SRIF) . A culture system for dispersed porcine pituitary cells was validated . Pituitaries from female pigs of various ages (90 or 110 d of gestation, newborn, 3, 6, or 24 mo old) were enzymatically dispersed with collagenase and neuraminidase, plated (200,000 cells/well), and cultured for 3 d . Plated cells were then subjected to a 4-h challenge with increasing concentrations of GRF (10(-11) to 10(-8) M), SRIF (10(-9) to 10(-6) M), or 10(-8) M of each peptide with increasing concentrations of the other . Culture media were collected and assayed for growth hormone (GH) . Pituitaries were pooled so that there were four replicates per age, and treatments were assigned to quadruplicate wells . Concentrations of GH in control wells (basal GH) were maximal at 110 d of gestation and decreased thereafter (P < .01) with increasing age of swine . All peptide combinations affected the GH response (P < .05) at all ages studied, yet GRF was more potent than SRIF in eliciting a response . Age had an effect (P < .05) on the GH response to any of the treatments; younger pigs (90, 110 d of gestation and newborns) had a greater response (P < .05) than older pigs (3, 6, and 24 mo), whereas 6- and 24-mo-old pigs responded similarly in all cases (P > .1).(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Reprod, 1993 Mar, 8(3), 364 - 8
Sandostatin has no direct effect on ovarian steroidogenesis in vitro; Pawelczyk LA et al.; Rat granulosa and theca-interstitial cells from immature, oestradiol-treated rats were isolated and incubated for 144 h with follicle stimulating hormone (FSH), luteinizing hormone (LH), insulin alone or in combinations, and with two doses of sandostatin (10(-7) M and 10(-6) M per culture) . Oestradiol and testosterone production by granulosa and theca-interstitial cells, respectively, was measured in culture media . The stimulatory effects of FSH alone and FSH with insulin but not insulin alone on oestradiol production by granulosa cells were observed . Similarly, increased testosterone concentrations after treatment with LH alone and LH with insulin but not insulin alone were found in media from theca-interstitial cells . The addition of high or low doses of sandostatin to the cultures did not affect the production of oestradiol or testosterone . It was concluded that sandostatin does not exert any direct effect on ovarian steroidogenesis in vitro.

J Clin Invest, 1993 Mar, 91(3), 838 - 44
Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism; Edery M et al.; A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism . The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor . All constructs were transiently expressed in COS-7 cells . Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively . Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities . In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand . These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients.

J Virol, 1993 Mar, 67(3), 1647 - 52
Calcium ions are required for cell fusion mediated by the CD4-human immunodeficiency virus type 1 envelope glycoprotein interaction; Dimitrov DS et al.; Calcium ions are required for fusion of a wide variety of artificial and biological membranes . To examine the role of calcium ions for cell fusion mediated by interactions between CD4 and the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41), we used two experimental systems: (i) cells expressing gp120-gp41 and its receptor CD4, both encoded by recombinant vaccinia viruses, and (ii) chronically infected cells producing low levels of HIV-1 . Fusion was measured by counting the number of syncytia and by monitoring the redistribution of fluorescence dyes by video microscopy . Syncytia did not form in solutions without calcium ions . Addition of calcium ions partially restored the formation of syncytia . EDTA and EGTA {ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid} blocked syncytium formation in culture media containing calcium ions . Membrane fusion as monitored by fluorescence dye redistribution also required calcium ions . Cell fusion increased with an increase in calcium ion concentration from 100 microM to 10 mM but was not affected by magnesium ions in the concentration range from 0 to 30 mM . Fibrinogen and fibronectin did not promote fusion in the absence or presence of Ca2+ . Binding of soluble CD4 to gp120-gp41-expressing cells was not affected by Ca2+ and Mg2+ . We conclude that Ca2+ is involved in postbinding steps in cell fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction.

Diabetes, 1993 Mar, 42(3), 411 - 9
Diabetes and embryonic malformations . Role of substrate-induced free-oxygen radical production for dysmorphogenesis in cultured rat embryos; Eriksson UJ et al.; The aim of this study was to evaluate the role of free-oxygen radicals in the embryonic maldevelopment of diabetic pregnancy . Rat embryos cultured in vitro during early organogenesis showed growth retardation and severe malformations after exposure to 50 mM glucose, 3 mM PYR, 10 mM HBT, or 3 mM KIC . Combinations of 25 mM glucose, 2.5 mM HBT, and 1 mM KIC also elicited embryonic growth retardation and malformations . The deleterious effects on embryonic development by all agents were alleviated by addition of SOD to the culture media, which yielded increased enzyme activity in the embryos and their membranes . The endogenous SOD activity also increased in embryos subjected to a high concentration of glucose or PYR in the culture medium . Addition of the mitochondrial PYR transport inhibitor CHC to the culture media blocked the dysmorphogenesis caused by glucose and PYR, but was without effect on the teratogenic actions of HBT and KIC . These findings implicate the embryonic mitochondria as a likely site for enhanced substrate-induced production of free-oxygen radicals mediating the teratogenic effect of a diabetic environment . In particular, the teratogenic process in diabetic pregnancy may depend on an increased production of free-oxygen radicals in immature embryonic mitochondria in response to a metabolic overload . This notion implies that every oxidative substrate entering the mitochondrial metabolism in excess may induce embryonic malformations and emphasizes the need for an extended metabolic surveillance of pregnant diabetic women . Consequently, optimal metabolic control should aim at normalizing the maternal serum concentrations of all possible oxidative substrates.

J Reprod Fertil, 1993 Mar, 97(2), 441 - 50
Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa; Aitken RJ et al.; The reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide (O2-.) and hydrogen peroxide (H2O2), respectively . With the aid of this system, the direct effect of reactive oxygen species (ROS) on human sperm function has been investigated . A protocol involving the addition of xanthine oxidase to the reaction mixture at 0 and 15 min resulted in a loss of motility involving every component of sperm movement examined . Lower doses of xanthine oxidase, which did not influence sperm motility, were also found to suppress the competence of human spermatozoa to exhibit oocyte fusion in response to the ionophore, A23187 . The reactive oxygen species responsible for the disruption of human sperm function was not influenced by the presence of superoxide dismutase (SOD) or scavengers of hypochlorous acid or hydroxyl radicals . However, the cytotoxic species was shown to be extremely stable and could be completely eliminated by catalase, which selectively eliminates H2O2 . Confirmation that it is H2O2, and not O2-., which is cytotoxic to human spermatozoa was obtained in studies in which the direct addition of this oxidant was shown to influence both the movement of human spermatozoa and their competence for oocyte fusion . These results carry implications for the diagnosis of defective sperm function and the design of optimized culture media for the treatment of male factor infertility.

J Hypertens, 1993 Mar, 11(3), 245 - 51
Production of polyclonal antisera to parathyroid hypertensive factor from spontaneously hypertensive rats; Benishin CG et al.; OBJECTIVE: Parathyroid hypertensive factor (PHF) is a newly described hypertensive factor which may cause elevation of blood pressure in approximately 40% of North American essential hypertensive patients . PHF is also found in several animal models of hypertension, including spontaneously hypertensive rats (SHR), deoxycorticosterone acetate-salt hypertensive rats and salt-sensitive Dahl rats . The objective of the present study was to raise an antibody to PHF, and to use this antibody to study the effect of PHF in SHR . DESIGN: Plasma and parathyroid gland culture media collected from SHR were used in the present study as the antigen in the production and analysis of a polyclonal antiserum to PHF . METHODS: PHF is of low molecular weight (approximately 3000 daltons) and is sensitive to inactivation by trypsin . We used PHF partially purified from parathyroid gland culture media as the antigen to inoculate mice . The substance was bound to aminophenyl thioether paper discs to make it more antigenic . The discs were then emulsified in Freund's adjuvant (complete for first inoculation, incomplete for subsequent inoculations) for intraperitoneal implantation . Production of anti-PHF antisera was monitored by enzyme-linked immunosorbent assay . RESULTS: Antisera produced in mice reacted with purified PHF prepared from SHR plasma as well as with PHF prepared from parathyroid gland culture media . PHF treated with the PHF antiserum produced no characteristic hypertensive response in normotensive assay rats . The antisera did not crossreact with two forms of bovine parathyroid hormone, bPTH (1-84), bPTH (1-34) or with shorter parathyroid hormone fragments, or with any other vasoactive substance tested . Injection of an aliquot of the antiserum in anesthesized spontaneously hypertensive rats reduced mean arterial pressure to a normal range of approximately 110 mmHg . CONCLUSIONS: These results indicate that (1) polyclonal antisera to PHF can be raised in mice and (2) PHF may contribute significantly to the elevated blood pressure in SHR.

In Vitro Cell Dev Biol, 1993 Mar, 29A(3 Pt 1), 239 - 48
Characterization of the DiFi rectal carcinoma cell line derived from a familial adenomatous polyposis patient; Olive M et al.; The DiFi human colorectal cancer cell line was recently established from a familial adenomatous polyposis patient with extracolonic features characteristic of the Gardner syndrome . These cells have now been propagated for 150 passages in standard culture media and vessels without feeder layers or collagen coatings . They retain features of colonic epithelial cells such as surface microvilli, secretory vesicles, and desmosomes . Cytosol of DiFi cells contains a high level (502 U/mg protein) of the mucin CA 19-9 . In addition, DiFi cells produce carcinoembryonic antigen, and induce tumors in athymic mice . Cytoskeleton analysis of DiFi cells by fluorescence microscopy showed a pronounced disorganization of actin cable structure . The isozyme genetic signature of DiFi cells is unique (0.01 probability of finding the same genetic signature in a different cell line), differs from that of HeLa cells, and has expressional features seen in other colorectal cell lines . The DiFi cell karyotype is tetraploid, contains many marker chromosomes, and shows numerous episomal particles . Two copies of chromosome 18 were absent, and only a single normal chromosome 17 was found . This parallels detection of allelic losses from DiFi cell DNA at loci on chromosomes 17p and 18 using molecular (cDNA) probes . DiFi cells clearly express transcripts for the c-myc proto-oncogene, the c-myb proto-oncogene, and the p53 tumor suppressor gene . Transforming growth factor beta inhibits DiFi cell growth in soft agar and suppresses c-myc expression in these cells . The value of this cell line in the study of genetic alterations in colorectal cancer is discussed.

J Neurosci Res, 1993 Mar 1, 34(4), 382 - 93
FGF-2-mediated protection of cultured mesencephalic dopaminergic neurons against MPTP and MPP+: specificity and impact of culture conditions, non-dopaminergic neurons, and astroglial cells; Otto D et al.; The protective role of basic fibroblast growth factor (FGF-2) for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and methylpyridiniumion (MPP+)-lesioned dopaminergic (DAergic) nigrostriatal neurons was studied, using dissociated cell cultures of embryonic day (E) 14 rat mesencephalon . Cells were grown in different culture media and received FGF-2 (5 ng/ml) and/or the toxins (5 microM) at various schedules, but were consistently allowed to differentiate for 3 days prior to becoming exposed to the toxin . Survival of tyrosine hydroxylase (TH)-immunoreactive cells at 7 days was only markedly impaired by MPTP, if horse serum (HS) or bovine serum albumin (BSA) were omitted from the culture medium . FGF-2 increased the number of TH-immunoreactive cells, and this increase was not diminished by MPTP under any culture condition . Uptake of 3H-DA was significantly reduced by MPTP in HS- and BSA-containing, but not in protein-less cultures . A protective effect by FGF-2 was only seen in the presence of BSA . MPP+ caused a more pronounced reduction in 3H-DA uptake than MPTP, and this effect was partially reversed by the addition of FGF-2, unless cultures contained HS . Neurofilament protein (NF), and indirect measure for the total number of neurons present in the cultures, was not significantly reduced by MPTP or MPP+ corroborating the specificity of the toxin for DAergic neurons, which constitute only a minor fraction in these cultures . In line with the wide spectrum of target neurons of FGF-2, this factor significantly increased NF contents under any culture condition . Quantification of the amounts of glial fibrillary acidic protein (GFAP) revealed stimulatory effects of FGF-2 (2.5- to 4-fold) and at least 10-fold higher levels in the presence as compared to the absence of HS . These data show that FGF-2 can protect DAergic neurons against MPTP- and MPP(+)-mediated damage . However, the effects of the toxins as well as of FGF-2 are partially dependent on culture conditions . Variations in the effectiveness of toxins and FGF-2 are not overtly related to the total numbers of neurons or astroglial cells, but may reflect culture type-dependent alterations of neuronal and glial metabolism.

Endocr Regul, 1993 Mar, 27(1), 11 - 5
Deregulated production of interleukin-8 (IL-8) in autoimmune thyroid disease studied by newly developed IL-8 radioimmunoassay; Hirooka Y et al.; We have developed a sensitive, reproducible and specific radioimmunoassay for human interleukin-8 (IL-8) . Using 125I-labelled IL-8 and polyclonal rabbit antisera raised against recombinant human IL-8, a competitive inhibition assay was developed which could detect 5 pg/ml of human IL-8 . Other interleukins, growth factors, hormones, peptides and lectins did not affect the assay . IL-8 measured in supernatants of culture media of stimulated human peripheral blood mononuclear cells (PBMC) . Kinetics of IL-8 production in SAC-stimulated PBMC from seven normal subjects revealed that the production of IL-8 was detectable within 12 h and reached a plateau at 24 h . IL-8 levels in SAC-stimulation of PBMC from untreated patients with autoimmune thyroid diseases (Graves' disease and chronic thyroiditis) were significantly higher than in normal controls . However, after treatment, IL-8 production decreased to normal . The present study demonstrates the usefulness of quantitating human IL-8 produced by PBMC and the presence of a deregulated production of IL-8 in autoimmune thyroid diseases.

Anticancer Res, 1993 Mar-Apr, 13(2), 481 - 6
Urokinase-type plasminogen activator: a paracrine factor regulating the bioavailability of IGFs in PA-III cell-induced osteoblastic metastases; Koutsilieris M et al.; The transplantation of PA-III rat prostate cancer cells onto rat skeleton produces osteoblastic metastases . Therefore w e studied the paracrine interactions between the PA-III cells and osteoblast-derived osteosarcoma cells (UMR 106 cells) . A serine protease secreted by PA-III cells hydrolyzed IGF-binding protein-1 and IGF-binding protein-2 (IGFBP-1 and IGFBP-2) detected in the cell culture media (CM) of OMR 106 cells by western ligand blotting . The serine protease of PA-III cell CM was purified using a benzamidine affinity column . This protease was a protein of 45-50 kDa on polyacrylamide gel electrophoresis under non-reducing conditions but generated two protein bands under reducing conditions; a) one of 33-35 kDa possessing protease activity and b) another of 20-25 kDa which was proteinolytically inactive . Sequence analysis identified the amino acid sequence of the a-chain (20-25 kDa band) and of the b-chain (33-35 kDa band) of rat urokinase-type plasminogen activator molecule . Urokinase purified from PA-III cell CM hydrolyzed IGFBPs of UMR 106 cells and stimulated the proliferation of UMR 106 cells in serum-free cultures . Its protease activity was abolished by benzamidine and aprotinin . Its mitogenic activity for osteoblasts was inhibited by anti-IGF-I monoclonal antibody . Northern blot analysis documented the expression of the urokinase-type plasminogen activator gene in the mRNA extracted from PA-III cells . Urokinase expression was inhibited by dexamethasone . Therefore, we conclude that urokinase-type plasminogen activator stimulates osteoblasts via an IGF-I dependent mechanism . Hydrolysis of the IGFBOPs at the sites of PA-III cell-induced bone tumors account for an increased bioavailability of IGFs . This may facilitate the development and the growth of PA-III cell-induced bone tumor and can also mediate the subsequent local osteoblastic reaction.

Growth Regul, 1993 Mar, 3(1), 34 - 7
Phosphorylation of insulin-like growth factor-binding protein-1 from different sources; Koistinen R et al.; During purification, insulin-like growth factor binding protein-1 from amniotic fluid was separated into five different peaks by anion exchange chromatography . These peaks represent differently phosphorylated forms of IGFBP-1 . The major peak (peak 1) is non-phosphorylated . Peaks 3, 4, and 5 are more phosphorylated and, in native polyacrylamide gel electrophoresis (PAGE), they migrate faster than peaks 1 and 2 . The more phosphorylated forms have higher IGF-I-binding affinity . Both dephosphorylated and phosphorylated peaks enhanced IGF-I stimulated DNA-synthesis in fetal skin fibroblast cell culture . They, however, inhibited the binding of IGF-I to the same cells . The phosphorylation of IGFBP-1 was changed during pregnancy . In decidua and in amniotic fluid the degree of phosphorylation increased from early to late pregnancy, as indicated by faster mobility of IGFBP-1 in native PAGE and increased relative amount of the more phosphorylated peaks in anion exchange chromatography . Human ovarian follicular fluid, culture media from human granulosa cells and endometrial adenocarcinoma cells (HEC-1-B) consisted mostly of the non-phosphorylated form of IGFBP-1.

J Immunol Methods, 1993 Feb 26, 159(1-2), 125 - 9
Purification of monoclonal antibodies from tissue culture medium depleted of IgG; Darby CR et al.; A method is described that allows rapid purification of IgG monoclonal antibodies from tissue culture supernatant; it is easy, relatively cheap and produces a product of high purity . Protein G linked to Sepharose fast flow gel has a high capacity for binding IgG and permits large volumes of supernatant to be loaded at high flow rates . However, Protein G cannot discriminate between the monoclonal antibody and other irrelevant IgG e.g . bovine IgG present in the tissue culture supernatant . Therefore, bovine IgG was removed from foetal calf serum by passage over protein G before being used as part of standard tissue culture medium . Hybridomas producing a rat anti-mouse (e.g . KT6) and a mouse anti-mouse (e.g . KJ23a) mAb were cultured in such tissue culture supernatant depleted of bovine IgG . Subsequent passage of the spent culture supernatants over protein G produced good yields of mAb of high purity and concentration . This method can been used to produce many different mAbs without the need for growing cells in a live host as ascites, or the need for specialised tissue culture media or expensive chromatography equipment.

J Biol Chem, 1993 Feb 25, 268(6), 4382 - 90
Stoichiometric analysis of internalization, recycling, and redistribution of photoaffinity-labeled guanylate cyclase/atrial natriuretic factor receptors in cultured murine Leydig tumor cells; Pandey KN; The membrane-bound form of guanylate cyclase represents a biologically active atrial natriuretic factor receptor (GC/ANF-R) . Murine Leydig tumor (MA-10) cells predominantly overexpress GC/ANF-R in high density (Pandey, K . N., Pavlou, S . N., and Inagami, T . (1988) J . Biol . Chem . 263, 13406-13413; Pandey, K . N., and Singh, S . (1990) J . Biol . Chem . 265, 12342-12348) . Information regarding the post-binding events of GC/ANF-R is obscure . This study presents the kinetics of internalization, recycling, and redistribution of GC/ANF-R in model MA-10 cells . Both the 125I-ANF binding assays and photoaffinity labeling procedures were utilized to label the total, intracellular, and cell surface GC/ANF-R . After the binding of 125I-ANF to GC/ANF-R, this complex was internalized and both the intact and degraded ligands were released into culture media . The distribution of 125I-ANF on the cell surface, in the intracellular compartments, and into culture media provided a dynamic relationship between the rates of 125I-ANF uptake, its degradation, and extrusion . The extent of receptor recycling was measured using tryptic proteolysis of photoaffinity-labeled GC/ANF-R to distinguish cell surface receptors from those that were internalized . A population of GC/ANF-R rapidly recycled (t1/2 = 5 min) from intracellular compartment to plasma membrane . Recycling of GC/ANF-R was impaired by chloroquine, dinitrophenol, and low temperature (22 degrees C) . Furthermore, these studies suggest that dissociation of ANF from the receptor is not required for recycling of internalized GC/ANF-R.

Int J Cancer, 1993 Feb 20, 53(4), 689 - 95
Expression of autocrine motility factor receptor in serum- and protein-independent fibrosarcoma cells: implications for autonomy in tumor-cell motility and metastasis; Watanabe H et al.; The motile response of serum-dependent (Gc-4 SD) and protein-independent (Gc-4 PF) murine fibrosarcoma cells to monoclonal antibody (MAb) that binds to gp78 a cell-surface receptor (M(r) 78,000) for an autocrine motility factor (AMF) was analyzed . The Gc-4 PF cells responded to the anti-gp78 by increased motility in vitro (3-fold) and increased lung colonization in vivo (8- to 20-fold), while the serum-dependent counterpart failed to respond to motile stimulation both in vitro and in vivo . Immuno-analysis of cell-surface expression and cell extracts revealed a smaller amount of gp78 in Gc-4 SD cells than in Gc-4 PF cells . Both cell lines secrete an equal amount of AMF to the culture media . Our results suggest that protein-free culture of Gc-4 PF cells is associated with high response to AMF and with high expression of its receptor, and that autonomous motile regulation may play a role in tumor dissemination.

Thromb Res, 1993 Feb 15, 69(4), 387 - 93
Construction and its expression of a new retroviral vector containing a human blood coagulation factor IX cDNA; Matsushita T et al.; We constructed a new factor IX expression vector containing a full length factor IX cDNA . The Moloney Murine Leukemia Virus (MoMLV)-based retroviral vector pLRNL was cloned with the entire coding region of human factor IX down stream of the promoter of Rous sarcoma virus, giving rise to the construct pL9RNL . The GP+E 86 packaging cell was transfected with pL9RNL and two mouse fibroblast cell lines (PA317 and NIH3T3) were infected with the virus generated from PA317 cell . During the 24 hr culture period, the maximum 1.2 micrograms of factor IX was secreted into the medium from 10(6) of GP+E 86 cells . Factor IX in the culture media had the relatively normal procoagulant activity and was barium-citrate precipitated . Western blotting analysis of the barium-citrate precipitates revealed that a single chain 50Kd protein indistinguishable from the plasma-derived factor IX was produced in these three cells . The retroviral expression system that we utilized herein may contribute to the study of recombinant wild type or various mutant factor IX, and to the basic study for the future gene therapy.

Nippon Ganka Gakkai Zasshi, 1993 Feb, 97(2), 156 - 61
{Induction of prostaglandin E2 by interleukin 1 in human lens epithelial cells}; Nishi K et al.; The authors proposed the hypothesis that pseudophakic inflammation, including the fibrin reaction, may be caused by interleukin (IL)-1, IL-6, other cytokines and/or prostaglandins (PGs), synthesized by residual lens epithelial cells (LEC) . In testing our hypothesis, we have already detected IL-1 alpha, IL-6 and PGE2 in the culture media of human LEC obtained by capsulotomy during cataract surgery . In this paper, we studied the time course of PGE2-synthesis and interaction between IL-1 and PGE2 . PGE2 concentration was measured by radioimmunoassay in the culture media to which rabbit antihuman IL-1 polyclonal antibody was added after culture for 6 h, 1 day, 1, 2, 3, 4, 5 and 6 weeks . There was no increase of PGE2, whereas in the untreated control culture media significant increase of PGE2 was confirmed . The results show that the antihuman IL-1 polyclonal antibody suppressed PGE2-synthesis . Thus, IL-1 induces PGE2 synthesis in human LEC culture.

Hum Reprod, 1993 Feb, 8(2), 288 - 95
Mouse embryo cleavage, metabolism and viability: role of medium composition; Gardner DK et al.; The cleavage, metabolism and viability of mouse zygotes were assessed after culture in media of different ionic and metabolite composition . Medium with a high potassium concentration, characteristic of mammalian oviduct fluid, inhibited cleavage and blastocyst formation (P < 0.01) . This inhibition was partially alleviated by the removal of phosphate, and subsequently abolished by supplementation with amino acids, vitamins, insulin, epidermal growth factor and transferrin (AVIET) . Glucose uptake by cultured blastocysts, measured fluorimetrically, was not affected by the ionic or metabolite composition of the medium, but was significantly reduced by the inclusion of AVIET (P < 0.01) . Lactate production was also significantly reduced in the presence of AVIET (P < 0.01) . Calculations of metabolic activity revealed that embryos cultured in the presence of AVIET had a glycolytic activity similar to embryos developed in vivo . In contrast, embryos cultured in conventional embryo culture media exhibited an elevated glycolytic activity . Culturing embryos for 4 days in a reduced lactate concentration (4.79 mM), significantly increased fetal development after transfer, compared with embryos cultured in the concentration of lactate present in conventional embryo culture media (23.3 mM; P < 0.01) . In contrast, when embryos were transferred on day 3 of culture, significantly more fetuses were obtained from embryos cultured in high levels of lactate (P < 0.01) . Supplementation of medium with AVIET significantly increased resultant fetal weights after transfer (P < 0.05) . This study demonstrates that different media are required to maintain embryo viability on successive days of culture, and highlights the potential limitations of employing simple salt solutions for the culture of preimplantation mammalian embryos.

Hum Reprod, 1993 Feb, 8(2), 266 - 71
Development of embryos from natural cycle in-vitro fertilization: impact of medium type and female infertility factors; Monks NJ et al.; Single embryos derived from natural cycle in-vitro fertilization (IVF) were graded during the pre-transfer culture period using morphological criteria . Most embryos developed well in culture with 96% showing continuing division and 68% showing good morphological appearance, although embryo quality tended to decline with an increased incidence of fragmentation and uneven cleavage as division proceeded . Both the pregnancy rate and the distribution of embryo grades were similar among four different culture media used, suggesting that choice of medium had little impact on outcome . In contrast, there were marked differences in pregnancy rate according to the type of infertility, which was not reflected in a decrease in embryo quality . However, although embryos from patients with tubal infertility implanted and formed viable pregnancies irrespective of morphological appearance, only 'good' quality embryos from patients with non-tubal (or 'unexplained') infertility were able to implant . Thus the appearance of the embryo derived from natural cycle IVF in women with unexplained infertility may be of clinical relevance.

Exp Eye Res, 1993 Feb, 56(2), 157 - 65
A soluble product of human corneal fibroblasts inhibits lymphocyte activation . Enhancement by interferon-gamma; Donnelly JJ et al.; Corneal stromal fibroblasts expressed HLA-DP, -DQ and -DR Class II MHC antigens in response to interferon-gamma, but did not induce proliferative responses by allogeneic peripheral blood mononuclear cells in vitro when used as stimulator cells in a mixed leukocyte-type reaction . Furthermore, corneal stromal fibroblasts inhibited mixed leukocyte reactions between peripheral blood mononuclear cells of allogeneic donors, even when the corneal stromal fibroblasts were separated from the peripheral blood mononuclear cells by a 0.4 micron pore membrane . Pretreatment of the corneal stromal fibroblasts with interferon-gamma increased the inhibitory activity . Both {3H}thymidine incorporation and interleukin-2 production were inhibited, and the inhibition appeared to be mediated by a soluble factor whose production required protein synthesis . The inhibitory activity was not abolished by including 1-10 micrograms ml-1 indomethacin in the culture media . No inhibition was observed in the proliferation dose-response curves of responder peripheral blood mononuclear cells that had been cultured with corneal stromal fibroblasts for 3 days, prior to culture with allogeneic stimulator peripheral blood mononuclear cells . Thus, the ability of corneal stromal fibroblasts to interfere with alloimmune responses in vitro was dependent upon the continued presence of the fibroblasts and their continued production of a soluble inhibitory factor or factors . Inhibitors of allogeneic reactions that are produced by corneal stromal fibroblasts stimulated by immune cytokines (e.g . interferon-gamma) may play a role in prolonging corneal allograft survival.

Blood Coagul Fibrinolysis, 1993 Feb, 4(1), 55 - 9
Purification and characterization of recombinant human fibrinogen; Lord ST et al.; Our laboratory has been using protein engineering to study the relationship of primary structure to fibrinogen function . In order to examine genetically altered domains in the context of the intact, functional fibrinogen molecule, we have expressed recombinant human fibrinogen in Chinese Hamster Ovary (CHO) cells . The cDNA for each fibrinogen chain was individually cloned into the same expression vector . Each vector was cotransfected with the selection vector pRSVneo into CHO cells . In addition, the plasmids encoding A alpha and gamma were cotransfected with pRSVneo . Cells resistant to G418, a neomycin analogue, were isolated and clonal lines developed . Analysis of these lines demonstrated that CHO cells express and secrete free gamma chain, and an A alpha-gamma complex . To obtain recombinant fibrinogen, the A alpha-gamma G418-resistant clones were transfected with the B beta expression plasmid and a second selection vector, pMSVhis . Colonies resistant to neomycin and histidinol were selected and clonal lines obtained . These clones secreted biologically active recombinant human fibrinogen, which was purified from serum-free culture media by protamine-Sepharose chromatography . Analysis of the purified protein on SDS-polyacrylamide gels demonstrated a pattern indistinguishable from plasma fibrinogen . Removal of Asn-linked carbohydrate with glycosidase F revealed the presence of carbohydrate on the B beta and gamma chains, as is seen for plasma fibrinogen.

Food Chem Toxicol, 1993 Feb, 31(2), 137 - 47
Culture of precision-cut liver slices: effect of some peroxisome proliferators; Beamand JA et al.; Precision-cut rat liver slices were prepared with a Krumdieck tissue slicer and cultured in three standard hepatocyte culture media . Rat liver slices cultured in either RPMI 1640 medium or Williams Medium E could be maintained in culture for up to 72 hr . In contrast, Leibovitz's L-15 medium was unsatisfactory in that slice viability, assessed either by morphological examination or by measurement of enzyme activities, could not be maintained for periods greater than 24 hr . As a measure of functional viability liver slices were cultured with some known rodent peroxisome proliferators, namely clofibric acid, nafenopin, ciprofibrate and Wy-14,643 . The peroxisome proliferators induced both palmitoyl CoA oxidation and carnitine acetyltransferase activities in 48- and 72-hr slice cultures . Ultrastructural examination of liver slices cultured with either ciprofibrate or Wy-14,643 for 72 hr revealed an increase in the number of peroxisomes . These results demonstrate that rat liver slices may be maintained in culture for up to 72 hr, and that they respond in a similar manner to rat primary hepatocyte cultures to some peroxisome proliferators . Precision-cut liver slices may therefore be a useful alternative in vitro system to hepatocyte cultures for screening compounds for effects on enzyme activities and for assessing species differences in response.

Am J Physiol, 1993 Feb, 264(2 Pt 2), H617 - 24
Plasmin potentiates induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells; Durante W et al.; Experiments were performed to examine the effect of the major fibrinolytic protease, plasmin, on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured human and rat aortic smooth muscle cells . Incubation of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite and nitrate in the culture media . Plasmin, either added exogenously or generated by the reaction of tissue plasminogen activator with plasminogen, potentiated the IL-1 beta-mediated release of nitrite and nitrate from smooth muscle cells in a concentration-dependent manner, without affecting the production of nitrite and nitrate from cells untreated with IL-1 beta . This potentiating effect was abolished when plasmin was incubated with the protease inhibitor, alpha 2-antiplasmin . The perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium, and the addition of IL-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated platelets inhibited their aggregation . Untreated smooth muscle cells or cells treated with plasmin alone did not have such effects . However, the simultaneous treatment of smooth muscle cells with IL-1 beta and plasmin markedly enhanced both the relaxing activities of the perfusates and the inhibition of platelet aggregation . Treatment of smooth muscle cells with NG-nitro-L-arginine inhibited the cytokine-mediated effects as well as the potentiating effect of plasmin . These results demonstrate that the plasmin can enhance the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells.

Am J Physiol, 1993 Feb, 264(2 Pt 2), H520 - 5
Dextran increases survival of subconfluent endothelial cells exposed to shear stress; Wechezak AR et al.; In vitro devices in combination with cultured cells have been used to study the relationship between shear stress and endothelial injury . Almost exclusively, these investigations have used confluent monolayers and conventional culture media as perfusates and reported little cell loss over a wide range of shear stress conditions . In this investigation when subconfluent endothelial cells were exposed to 22 and 88 dyn/cm2 for 2, 8, and 24 h in a perfusate of medium and 5% serum, a progressive cell loss was observed . Lower cell densities were a product of decreased cell proliferation as measured by bromodeoxyuridine (BrdU) incorporation and loss of the initial cell population . Video recordings indicated that cells characteristically detached by proximal cell peeling from the substrate and an aneurysmal rupture of the cell membrane . Cell retention was increased by including 250 and 475 microM neutral dextran (70 kDa) in perfusates . Experimental evidence suggests dextran does not directly stimulate proliferation or correct an osmotic imbalance . This investigation has substantiated that fluid-generated shear stress can cause endothelial denudation and that conditions (subconfluency, time, and perfusate supplementation) under which shear stress is applied are as important for cell survival as shear stress magnitude.

Int J Biochem, 1993 Feb, 25(2), 219 - 21
Effect of LCAT on HDL-mediated cholesterol efflux from loaded EA.hy 926 cells; Kilsdonk EP et al.; 1 . Human endothelial cells (EA.hy 926 line) were loaded with cholesterol, using cationized LDL, and the effect of lecithin:cholesterol acyltransferase (LCAT) on cellular cholesterol efflux mediated by high density lipoproteins (HDL) was measured subsequently . 2 . In plasma, lecithin:cholesterol acyltransferase (LCAT) converts unesterified HDL cholesterol into cholesteryl esters, thereby maintaining the low UC/PL ratio of HDL . It was tested if further decrease in UC/PL ratio of HDL by LCAT influences cellular cholesterol efflux in vitro . 3 . Efflux was measured as the decrease of cellular cholesterol after 24 hr of incubation with various concentrations of HDL in the presence and absence of LCAT . LCAT from human plasma (about 3000-fold purified) was added to the cell culture, resulting in activity levels in the culture media of 60-70% of human serum . 4 . Although LCAT had a profound effect on HDL structure (UC/TC and UC/PL ratio's decreased), the enzyme did not enhance efflux of cellular cholesterol, using a wide range of HDL concentrations (0.05-2.00 mg HDL protein/ml) . 5 . The data indicate that the extremely low unesterified cholesterol content of HDL, induced by LCAT, does not enhance efflux of cholesterol from loaded EA.hy 926 cells . It is concluded that the HDL composition (as isolated from plasma by ultracentrifugation) is optimal for uptake of cellular cholesterol.

J Parasitol, 1993 Feb, 79(1), 23 - 31
Proteinase activity in miracidia, transformation excretory-secretory products, and primary sporocysts of Schistosoma mansoni; Yoshino TP et al.; Proteinase activity was detected in the culture medium of transforming miracidia and in detergent extracts of Schistosoma mansoni miracidia and primary sporocysts using a fluorescent substrate, carbobenzoxy-phenylalanyl-arginyl-7-amino-4- trifluoromethylcoumarin . Medium collected after the first 24 hr of miracidial cultivation (transformation medium; TM) contained most (80%) of the activity released during 5 days of in vitro culture . Based on proteinase activity contained in Triton X-100 extracts of whole larvae, miriacidia and primary sporocysts exhibited a similar amount of total activity per organism, whereas specific activity was about 2-fold greater in miracidia . Approximately 10% of total miracidial activity was released during the first 24 hr of transformation . This early release of proteinase is consistent with possible involvement of these enzymes in miracidial snail penetration . Proteinase activities from larval extracts and culture media were identical when characterized for thiol-dependence, inhibitor profile, and pH optimum and indicate that the proteinase(s) belongs to the cysteine class of acidic endopeptidases . Further studies with TM revealed a substrate preference for a hydrophobic amino acid in the P2 position . High performance liquid chromatography gel filtration showed 2 peaks of activity at 19,000 and 36,000 Da, whereas specific inhibitor labeling yielded heterogeneous banding in the molecular weight range of 33,000-44,000 Da . Lastly, sporocyst extracts incubated with snail plasma (cell-free hemolymph) revealed degradation of high molecular weight hemolymph proteins, including hemoglobin . The finding of significant cysteine proteinase activity in miracidia and primary sporocysts and the continued low level of secretion by sporocysts suggest a functional role of these proteinases in the establishment and/or maintenance of infections within the snail host.

Bone Marrow Transplant, 1993 Feb, 11(2), 169 - 73
Polymerase chain reaction: a method for monitoring tumor cell purge by long-term culture in BCR/ABL positive acute lymphoblastic leukemia; Fabrega S et al.; We report the successful purging of leukemia cells bearing the Philadelphia chromosome and BCR/ABL transcripts by long-term marrow culture (LTC), and subsequent grafting of the purged marrow in a case of refractory acute lymphoblastic leukemia . The efficiency of the purge was evaluated by polymerase chain reaction (PCR) for BCR/ABL transcripts . In two LTCs initiated in the blastic stage, we demonstrated the selective effect of three culture media (serum dependent, serum-free (SF) supplemented or not with IL3 and GM-CSF) on the proliferative potential of normal hematopoietic (CFU-GM/BFU-E) and leukemic progenitors (CFU-ALL) . BCR/ABL positive cells disappeared after 3 to 4 weeks of culture . The addition of IL3 and GM-CSF to the SF medium enhanced the growth of CFU-GM/BFU-E and shortened the purging period . We therefore carried out a LTC in the presence of IL3 and GM-CSF with marrow harvested in morphological remission . BCR/ABL positivity was detected at the outset, although no leukemia cells could be identified . The BCR/ABL was no longer found by PCR in the 7 and 14 day LTCs . The patient, consolidated by high dose polychemotherapy and total body irradiation, was infused with the 14 day LTC . This study indicates that PCR is a useful and sensitive technique for monitoring tumor cell reduction after LTC prior to autografting.

Arch Ophthalmol, 1993 Feb, 111(2), 263 - 7
Prolonged localized tissue effects from 5-minute exposures to fluorouracil and mitomycin C; Khaw PT et al.; Rabbits undergoing full-thickness glaucoma filtering surgery were exposed for 5 minutes to one of three intraoperative treatments: (1) distilled water; (2) fluorouracil, 50 mg/mL; or (3) mitomycin C, 0.4 mg/mL . Tissue samples were taken from the subconjunctival and scleral tissues at the treated area and 90 degrees and 180 degrees from the center of the treated area and the adjacent cornea 2 mm from the limbus, 1 hour, 5 days, and 30 days postoperatively . The biopsy specimens were then placed in tissue culture media and the fibroblast outgrowths measured . Five-minute intraoperative treatments with fluorouracil resulted in a reversible delay of fibroblast outgrowths from treated subconjunctival and scleral tissues of just over 1 week in this model, whereas treatment with mitomycin C, 0.4 mg/mL, resulted in prolonged inhibition of at least 30 days . These effects were localized to the area treated . The many clinical implications of these findings are discussed.

Endocrinology, 1993 Feb, 132(2), 584 - 90
Androgens augment vasoactive intestinal peptide- and growth hormone-releasing hormone-stimulated progestin production by rat granulosa cells; Li YD et al.; Vasoactive intestinal peptide (VIP) has been shown to stimulate steroid production by cultured rat granulosa cells independently of FSH . In the present study, we have examined the modulatory effects of various steroids on this response . Rat granulosa cells cultured for 2 days with only VIP showed small but significant increases in progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production . Concomitant treatment with either a synthetic estrogen (diethylstilbestrol), a synthetic progestin (R5020), or cortisol did not augment VIP-stimulated progesterone production; however, the latter two steroids slightly, but significantly, augmented VIP-stimulated 20 alpha-OH-P production . In contrast, concomitant treatment with a synthetic androgen (R1881) dramatically augmented both VIP-stimulated progesterone and 20 alpha-OH-P production . These effects were dose dependent for both VIP and R1881 and could be blocked by the androgen antagonist cyproterone acetate . Time course studies revealed that progesterone content of the culture media rapidly increased over the first 24 h of culture then remained fairly constant for the next 48 h; 20 alpha-OH-P content, on the other hand, was low for the first 12 h of culture and steadily increased thereafter . Dose-response analysis for R1881 revealed an ED50 of approximately 2 x 10(-8) M for the synthetic androgen, and comparison with other naturally occurring androgens provided the rank order of potency R1881 > androstenedione > testosterone = dihydrotestosterone . Additional studies with another member of the VIP peptide family, GH-releasing hormone, showed dose-dependent stimulation of progesterone and 20 alpha-OH-P production by this peptide . These effects were also augmented by R1881 but not by diethylstilbestrol, R5020, or cortisol . These studies demonstrate that androgens, but not estrogens, progestins, or glucocorticoids, augment VIP- and GH-releasing hormone-stimulated progestin production by cultured rat granulosa cells.

Mol Cell Biol, 1993 Feb, 13(2), 841 - 51
Proto-oncogenes of the fos/jun family of transcription factors are positive regulators of myeloid differentiation; Lord KA et al.; The proto-oncogenes c-jun, junB, junD, and c-fos recently have been shown to encode for transcription factors with a leucine zipper that mediates dimerization to constitute active transcription factors; juns were shown to dimerize with each other and with c-fos, whereas fos was shown to dimerize only with juns . After birth, hematopoietic cells of the myeloid lineage, and some other terminally differentiated cell types, express high levels of c-fos . Still, the role of fos/jun transcription factors in normal myelopoiesis or in leukemogenesis has not been established . Recently, c-jun, junB, and junD were identified as myeloid differentiation primary response genes stably expressed following induction of terminal differentiation of myeloblastic leukemia M1 cells . Intriguingly, c-fos, though induced during normal myelopoiesis, was not induced upon M1 differentiation . To gain further insights into the role of fos/jun in normal myelopoiesis and leukemogenicity, M1fos and M1junB cell lines, which constitutively express c-fos and junB, respectively, were established . It was shown that enforced expression of c-fos, and to a lesser extent junB, in M1 cells results in both an increased propensity to differentiate and a reduction in the aggressiveness of the M1 leukemic phenotype . M1fos cells constitutively expressed immediate-early and late genetic markers of differentiated M1 cells . The in vitro differentiation of normal myeloblasts into mature macrophages and granulocytes, as well as the increased propensity of M1fos leukemic myeloblasts to be induced for terminal differentiation, was dramatically impaired with use of c-fos antisense oligomers in the culture media . Taken together, these observations show that the proto-oncogenes which encode for fos/jun transcription factors play important roles in promoting myeloid differentiation . The ability of the M1 leukemic myeloblasts to be induced for terminal differentiation in the absence of apparent fos expression indicates that there is some redundancy among the fos/jun family of transcription factors in promoting myeloid differentiation; however, juns alone cannot completely compensate for the lack of fos . Thus, genetic lesions affecting fos/jun expression may play a role in the development of "preleukemic" myelodysplastic syndromes and their further progression to leukemias.

Am J Physiol, 1993 Feb, 264(2 Pt 2), H441 - 7
Regulation of adenosine receptor system in coronary artery: functional studies and cAMP; Hussain T et al.; Regulation of the adenosine receptor system was investigated using a new in vitro model of porcine coronary artery after exposure to an adenosine receptor agonist . Isolated coronary arteries were incubated in the absence (control) and presence (treated) of 2-chloroadenosine (CAD) in culture media (pH 7.4) at 37 degrees C with 92.5% O2-7.5% CO2 . CAD treatment led to a significant rightward shift in the relaxation responses to the adenosine receptor agonists CAD, 5'-(N-ethylcarboxamido)adenosine, and R-phenylisopropyladenosine . The shift was both concentration and time dependent and was maximum at 100 microM at 3 days . The relaxation responses to isoproterenol and sodium nitroprusside were unaltered under identical conditions . Forskolin-mediated relaxation was also shifted rightward in the treated group . The shift in CAD- and forskolin-mediated relaxation in the treated group was endothelium independent . On the other hand, isoproterenol (10 microM, 3 days) treatment produced a rightward shift in the relaxation response to isoproterenol, while the response to CAD was unaltered . In a separate set of experiments, CAD did not stimulate adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in the treated arteries compared with control . However, NaF and forskolin stimulation of cAMP was markedly decreased in the treated group . Unlike these agonists, isoproterenol stimulation of cAMP was similar in both treated and control groups . The data suggest the desensitization of the adenosine receptor via regulation of stimulatory guanine nucleotide binding proteins without altering the beta-receptor-mediated responses in coronary artery.

J Clin Microbiol, 1993 Feb, 31(2), 175 - 8
Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis; Telenti A et al.; A method for the rapid identification of mycobacteria to the species level was developed on the basis of evaluation by the polymerase chain reaction (PCR) of the gene encoding for the 65-kDa protein . The method involves restriction enzyme analysis of PCR products obtained with primers common to all mycobacteria . Using two restriction enzymes, BstEII and HaeIII, medically relevant and other frequent laboratory isolates were differentiated to the species or subspecies level by PCR-restriction enzyme pattern analysis . PCR-restriction enzyme pattern analysis was performed on isolates (n = 330) from solid and fluid culture media, including BACTEC, or from frozen and lyophilized stocks . The procedure does not involve hybridization steps or the use of radioactivity and can be completed within 1 working day.

Zhonghua Bing Li Xue Za Zhi, 1993 Feb, 22(1), 25 - 9
{The inhibitory effect of antisense oligodeoxynucleotides on the growth of human pancreatic adenocarcinoma cell lines}; Liu TH; In order to study the inhibitory effect of antisense oligodeoxynucleotides on the growth of human pancreatic adenocarcinoma cell lines (PC-2 & PC-3), synthesized c-myc & c-Ki-ras antisense oligodeoxynucleotides (ASODN) and sense oligodeoxynucleotides (SODN) were added to the culture media of PC-2 & PC-3 cell lines . The cell growth activities after 24, 48, 72 hours of culture were estimated by cell count and 3H-TdR incorporation, and the concurrent oncogene expressions were studied by adopting reverse transcription PCR technique . Cell growth was significantly inhibited after exposure to ASODN for 24 & 48 hours . Cell growth in the c-Ki-ras ASODN group approached nearly the same level as in the control groups 72 hours after exposure, whereas cell proliferation in the c-myc ASODN medium was still being inhibited . By reverse transcription PCR technique, marked inhibition of c-myc & c-Ki-ras expression was seen in the groups after 24 & 48 hours treatment of c-myc & c-Ki-ras ASODNs . There was also down-regulation of c-myc & c-Ki-ras expression after 72 hours exposure to both oncogene ASODNs.

J Cell Sci, 1993 Feb, 104 ( Pt 2), 289 - 96
Induction of tenascin in cancer cells by interactions with embryonic mesenchyme mediated by a diffusible factor; Hiraiwa N et al.; Human cancer cell lines A431 and MCF7, which do not produce tenascin (TN) in vitro, were found to produce TN when injected into nude mice or co-cultured with the embryonic mesenchyme . The TN expression in the developing A431 solid tumor was demonstrated by immunohistochemistry and by in situ hybridization . Human TN was detected in culture media by western blot analysis using human specific monoclonal antibody (RCB-1) . During tumorigenesis, in the early stage, mouse TN was actively induced and deposited in the peri- and intertumor spaces surrounding the developing tumor . Two days later, TN derived from human epithelial cancer cells was induced and mainly deposited in the intertumor basement membrane . After this stage, tumor cells were actively producing TN . On the other hand, TN induction in non TN-producing cells, such as A431 and MCF7 cell lines, was also observed in vitro . Although cell lines such as NIH-3T3, phi 2, STO, 2H6, 3E5 and CMT315, had no effect on the TN induction, primary cultured embryonic mesenchyme effectively stimulated the TN expression in the cancer cell lines . This mesenchymal effect decreased with age and was entirely lost postnatally . Furthermore, conditioned media from these embryonic mesenchymes could reproduce the same effects on TN induction as observed in the co-culture study . In conclusion, these findings suggest that TN induction in epithelial cancer cells may depend on interactions with the surrounding environment, that these interactions may be mediated by a soluble factor(s) derived from the surrounding mesenchyme and that the TN induction observed in the tumorigenesis may reflect histogenesis during the embryonic period.

Nippon Rinsho, 1993 Feb, 51(2), 323 - 8
{Expression of hepatitis C virus genome}; Nishihara T et al.; Complementary DNAs from HCV genome were expressed and analyzed . C8-2 protein, a part of NS5, and core protein were synthesized in E . coli . Core protein (JCC) was appeared to be very useful for diagnostic probe of HCV infection . Predicted envelope glycoproteins (E1 and E2/NS1) produced in infected insect cells were glycosylated and secreted into the culture media when the signal sequence of rabies virus G protein was introduced . An ELISA was developed using each purified recombinant protein . Anti-E1 antibody was detected in 20% of patients with non-A, non-B chronic liver diseases, whereas anti-E2/NS1 antibody was detected in more than 88% . These results indicate that the immune responses against these glycoproteins are different.

J Endocrinol Invest, 1993 Feb, 16(2), 109 - 15
In vitro detection of glycoprotein production and secretion by human nonfunctioning pituitary adenomas; Saccomanno K et al.; This study, carried out on 9 nonfunctioning pituitary adenomas, was undertaken in order to evaluate the ability of these tumors to synthesize and release gonadotropins and/or free alpha-subunit (alpha-SU) of glycoproteins . The morphological study included electron microscopy and immunofluorescence analysis while hormone release was evaluated by the reverse hemolytic plaque assay (RHPA) and measurements in culture media . By electron microscopy in all tumors (6 null cell adenomas and 3 oncocytomas), it was possible to identify rough endoplasmic reticulum, Golgi apparatus and secretory granules . By immunofluorescence, 5 of 6 tumors were immunoreactive for one or more gonadotropin subunits; in particular, 5 adenomas were positive for alpha-SU and LH-beta, and 3 for FSH-beta . By the RHPA, about 1% of cells obtained from one single tumor formed plaques for LH-beta and alpha-SU while the remaining tumors were negative . Similarly, the study of media concentrations of LH, FSH and alpha-SU in 2 h culture revealed very low amounts of released hormones . In these experimental conditions no modification was observed after the addition of stimulatory agents such as TRH, GnRH and VIP . The present study clearly indicates that although the large majority of nonfunctioning tumors are positive for gonadotropins their secretory capacity is very low in both basal and stimulated conditions.

Glia, 1993 Feb, 7(2), 121 - 33
Characterization of "plasma proteins" secreted by cultured rat macroglial cells; Zahs KR et al.; The brain is isolated behind a blood-tissue barrier that restricts the access of circulating proteins to neural cells . There is evidence that some of these proteins are synthesized within the central nervous system . The present study examines the synthesis and secretion of such proteins by cultured macroglial cells . Primary glial cultures were derived from cortical and subcortical regions of neonatal rat brains, and subsequent secondary cultures were enriched in type-1 astrocytes, type-2 astrocytes, or oligodendrocytes . Newly synthesized proteins were immunoprecipitated from the culture media using antisera directed against whole rat serum . All three types of glial cells secreted a range of plasma proteins . In general, type-1 astrocytes secreted more of these proteins than did type-2 astrocytes or oligodendrocytes, although the one-dimensional polyacrylamide gel electrophoresis (PAGE) profiles were specific for each cell type . Anti-sera directed against specific plasma proteins identified three of the most abundant proteins secreted by type-1 astrocytes as transferrin, alpha-2-macroglobulin, and ceruloplasmin . Northern blot analysis of cellular RNA confirmed that type-1 astrocytes contained transferrin mRNA, and that it was more abundant in cultures derived from subcortical regions than from cortical regions . In situ hybridization studies revealed that virtually all type-1 and type-2 astrocytes contained transferrin mRNA . Since the proteins identified in this study have been proposed to have a variety of neurotrophic roles in the central nervous system, these data further extend the range of possible functions that glial cells may serve in the CNS.

Leukemia, 1993 Feb, 7(2), 172 - 6
Expression of the hyaluronan-binding glycoprotein hyaluronectin in leukemias; Delpech B et al.; Hyaluronectin (HN), a hyaluronan (hyaluronic acid, HA)-binding glycoprotein is normally expressed in the nervous system, found in the desmoplasia of tumours, and is also produced in vitro by peripheral blood mononuclear cells . We have therefore investigated the expression and the production of HN by leukemic cells, with the hypothesis that HN would be expressed in leukemias of the myeloid lineage . Fresh and frozen leukemic cells were studied from 70 patients of whom 53 had acute myeloblastic leukemia (AML) . HN was strongly expressed (> 80% blood cells) in two out of 13 M4 AMLs and four out of four M5B AMLs . One further M4 AML displayed 25% positive cells and two 20% cell positivity cases were seen, in one case of M4 AML and in one case of chronic myelomonocytic leukemia (CMML) . The rest of the cases of AML as well as all cases of acute lymphoblastic leukemia (ALL) showed almost no positivity (< 1%) . The residual positive cells appeared to be normal blood promonocytes . Taken together > or = 20% positive cells was seen in eight out of 56 (14%) examined myeloid leukemias . The HN production was significantly higher (p < 0.0001) in cell culture media of M4 and M5 AML cells than in other AML or ALL cell culture media . A significant correlation was found (p < 0.0001) between the number of HN-positive leukemic cells and the number of cells with a monocytic morphology, suggesting that HN is a marker for the promonocyte.

Cell, 1993 Jan 29, 72(2), 197 - 209
The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage; Nguyen HQ et al.; We have isolated cDNA clones of myeloid differentiation primary response (MyD) genes, activated in the absence of de novo protein synthesis following induction for differentiation along either the macrophage or granulocyte lineage in human myeloblastic leukemia HL-60 cells . One cDNA clone of a primary response gene, expressed upon macrophage differentiation, encoded for Egr-1, a zinc finger transcription factor . The Egr-1 gene was observed to be transcriptionally silent in HL-60 cells, but active in U-937 and M1 cells, the latter two being predetermined for macrophage differentiation . Egr-1 antisense oligomers in the culture media blocked macrophage differentiation in both myeloid leukemia cell lines and normal myeloblasts . HL-60 cells constitutively expressing an Egr-1 transgene (HL-60Egr-1) could be induced for macrophage, but not granulocyte, differentiation . These observations indicate that expression of Egr-1 is essential for and restricts differentiation of myeloblasts along the macrophage lineage.

Brain Res, 1993 Jan 22, 601(1-2), 213 - 20
Cultures of ensheathing cells from neonatal rat olfactory bulbs; Chuah MI et al.; We have derived highly enriched populations of ensheathing cells (ECs) from the olfactory nerve layer of neonatal rat olfactory bulbs . Contaminating cells, such as fibroblasts, were eliminated from EC cultures by cytosine arabinoside and immunoadsorption with antiserum to Thy-1.1 . At the same time, ECs were stimulated to divide by the addition of bovine pituitary extract into the culture media . Confluent cultures containing 96-99% ECs, were comprised of either spindly bipolar cells or cells bearing multiple processes oriented on opposite poles . The ECs immunostained positively for GFAP, S-100 protein, N-CAMs and Neu 5, and were negative for the presence of neurofilaments . Scanning and transmission electron microscopy showed that the ultrastructure of the ECs resembled that in vivo . The nucleus was irregular in shape, intermediate filaments were usually scattered throughout the cytoplasm instead of being grouped into bundles, and rough endoplasmic reticulum existed as isolated expanded profiles.

Am J Med Genet, 1993 Jan 15, 45(2), 246 - 51
Somatic cell mosaicism: another source of phenotypic heterogeneity in nuclear families with osteogenesis imperfecta; Constantinou-Deltas CD et al.; Mutations in the genes coding for the pro alpha 1 and pro alpha 2 chains of type I procollagen have been found in many patients with osteogenesis imperfecta (OI), a heritable disorder of connective tissue . The severity of the disease varies between families and even among members of the same family . This phenotypic variability covers a spectrum extending from very mild forms that cannot be easily detected to perinatally lethal forms . One explanation for this phenotypic variability is the nature of the mutation in the type I procollagen genes . Another explanation is mosaicism . Here we report on 2 families with propositi who have OI, whereas their mothers had a milder form of the disease . In one family, the molecular defect was previously shown to be a substitution of alpha 1(904) by cysteine {Constantinou et al., 1990} . The biochemical phenotype was characterized by significant post-translational overmodification of the mutated type 1 collagen molecules which also had a 3-4 degrees C decrease in their thermal unfolding . Also, secretion of the procollagen into the culture media was delayed . In the second family, the proposita's muscle fibroblasts synthesized and secreted type I procollagen molecules that were highly over-modified along the entire length of their triple-helical domain . Cells from the mother also synthesized normal and over-modified protein, although the amount of over-modified protein was less than that synthesized by her daughter's cells . The exact molecular defect has not yet been defined.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1993 Jan 15, 150(2), 367 - 76
Anergized T cell clones retain their cytolytic ability; Go C et al.; CD4+ T cells have been described to have both helper and lytic function . The helper function of Th1 cells in particular can be inactivated by inducing the T cell into a state of nonresponsiveness in which the T cell is no longer capable of producing IL-2 or proliferating in an autocrine way to a conventional antigenic stimulus . To determine whether the lytic ability of Th1 cells can also be rendered nonfunctional upon anergy induction, we induced Th1 clones into a nonresponsive state and tested their ability to lyse target cells in an Ag-specific and MHC class II-restricted manner . We show that cells newly induced into an anergic state were able to lyse target cells nonspecifically . This effect was short-lived and after resting in culture media, the cells regained their ability to lyse target cells in an Ag/MHC-specific manner, and this ability was comparable to normal resting T cells . In contrast, the helper function of these cells remained nonresponsive, and the cells were unable to proliferate or to secrete IL-2 in response to the same antigenic stimulus used for lysis . Therefore, the lytic pathway appears to be regulated separately from the proliferative/lymphokine pathway(s) and is not affected long-term by an anergic stimulus.

Life Sci, 1993, 52(24), 1977 - 84
Effects of specific fatty acids on prolactin-induced NB2 lymphoma cell proliferation; Sylvester PW et al.; Nb2 rat lymphoma cells are dependent on prolactin (PRL) for growth . Membrane lipid composition of Nb2 cells undergoes rapid modification when these cells are grown in culture media supplemented with specific fatty acids . Since the actions of PRL are mediated through specific membrane receptors, the following studies were conducted to characterize the lipid-dependent events involved in fatty acid modulation of PRL-induced cell proliferation . Nb2 cells were grown in suspension cultures in control or fatty acid-supplemented media, in the presence of various doses of PRL . PRL-induced cell growth was significantly enhanced by arachidonate, but significantly attenuated by stearate supplementation of the culture media . A direct relationship was observed between the concentration of specific fatty acid added to the culture media and the magnitude with which this fatty acid was incorporated into Nb2 cell membranes, as determined by gas chromatography . Acute treatment with phorbol ester enhanced Nb2 cell growth in control media and reversed the attenuating effects of membrane stearic acid enrichment . However, PRL-induced Nb2 cell growth was similar with or without the presence of phorbol ester, when cells were grown in media supplemented with arachidonate . Addition of protein kinase C (PKC) inhibitors to control and fatty acid-supplemented media resulted in a dose-dependent inhibition of PRL-induced Nb2 cell proliferation . These results suggest that lipid modulation of Nb2 mitogenic-responsiveness to PRL is mediated through alterations in PKC activation.

J Fr Ophtalmol, 1993, 16(2), 80 - 6
{Ultrastructural study of human cornea preserved in organ culture media at +31 degrees C}; Borderie V et al.; Organ culture becomes a standard method of corneal graft preservation . The aim of the study was to evaluate the preservation injuries induced by organ culture . We examined 12 organ cultured human corneas . Corneas were preserved in the medium for 2 to 21 days . Corneas showed some abnormalities: numerous light vacuoles, mitochondrial swelling and increased cell thickness in all cells of the cornea, sloughing of the external and medium epithelial cell layers . We observed normal endoplasmic reticulum, Golgi apparatus and nucleus . These preservation injuries are moderate and reversible.

J Diabetes Complications, 1993 Jan-Mar, 7(1), 44 - 8
Taurine prevents galactose-induced cataracts; Malone JI et al.; Intact lenses from New Zealand white rabbits were incubated in tissue culture media containing either 5 mM glucose or 5 mM glucose plus 30 mM galactose . The standard media did not contain taurine . Lenses were also cultured in a third medium containing 30 mM galactose plus 0.2 mM taurine . The frequency of cataract formation was evaluated as a function of the culture media . One lens (1/10), in media containing 5 mM glucose, developed a lenticular opacification during a 72-h incubation . Lenses (12/15) incubated in 30 mM galactose, without taurine, developed cataracts; fewer lenses (2/13) exposed to 30 mM galactose plus 0.2 mM taurine developed cataracts (p < 0.005) . Galactose cataracts have been associated with lens edema attributed to the osmotic stress of tissue polyol (galactitol) accumulation . The water content of the noncataractous and cataractous lenses in this experiment did not differ . Lens edema, therefore, was not thought to be important in cataract pathogenesis . Taurine, an organic osmolyte was lower (5.1 +/- 1.5 mumol/g protein) in cataractous lenses than in control lenses (10.0 +/- 1.0 mumol/g protein) . Malondialdehyde, an indicator of lipid peroxidation, was higher (36.6 +/- 5.0 mumol/g protein) in lens-containing opacifications than in noncataractous lenses (10.1 +/- 1.9 mumol/gm protein) (p < 0.01) . The levels of malondialdehyde suggest that lipid peroxidation was increased in the process of sugar cataractogenesis . The malondialdehyde content of all the lenses correlated inversely (r = -0.53, p < 0.01) with the coincident lens taurine levels . Taurine appears to protect the lens against the development of sugar cataracts; its inverse relationship with lens malondialdehyde suggests this is an antioxidant effect.

Anticancer Res, 1993 Jan-Feb, 13(1), 57 - 64
Cytotoxic activity of fusaric acid on human adenocarcinoma cells in tissue culture; Fernandez-Pol JA et al.; When cultured human normal WI-38 fibroblasts were exposed to 500 microM fusaric acid (5-butylpicolinic acid), cell proliferation ceases . The majority of the WI-38 cells remained in a quiescent G1(G0) state and viable for approximately 30 to 48 h . The effect of fusaric acid on WI-38 cell growth was slowly reversible after removal of the agent from the culture media . In contrast, within 24 h of exposure to 500 microM fusaric acid, most of the human colon adenocarcinoma LoVo cells were blocked at random in the cell cycle and approximately 80% of the cells were dead after 30 h . Three additional human colorectal adenocarcinoma cell lines, denoted SW48, SW480, and SW742, were also sensitive to the inhibitory and cytotoxic effects of fusaric acid . Of all the cell lines tested, the human mammary adenocarcinoma cell line MDA-MB-468 was the most sensitive to the growth inhibitory and cytotoxic effects of fusaric acid . Further experiments with MDA-MB-468 cells demonstrated that fusaric acid is a potent inhibitor of DNA synthesis . Fusaric acid also inhibited the growth of human epidermoid carcinoma KB cells and showed cytotoxic actions for this cell line but its effects on cell viability were not as pronounced . The results presented here indicate that fusaric acid has potent anti-proliferative activity in vitro on various normal and cancer cell lines and suggest that it exhibits some cytotoxic specificity for growing and confluent colorectal adenocarcinoma and mammary adenocarcinoma cell lines.

Nutrition, 1993 Jan-Feb, 9(1), 37 - 42
Amino acid imbalance and intracellular protein synthesis; Nishihira T et al.; The effects of amino acid imbalance, created by decreasing the concentrations of specific amino acids in culture media, on intracellular protein synthesis were compared with the findings obtained by restricting all amino acids . Protein-synthesis levels were estimated by measuring the uptake rates of 14C-labeled amino acids by cultured rat hepatoma AH109A cells . The inhibitory activity of a 0.1-fold dilution of valine in DM-160 medium was higher than that resulting from the dilution of all amino acids by 0.1-fold . On the other hand, the dilution of other essential amino acids was not as effective as the dilution of all amino acids, whereas the concurrent dilution of valine and leucine was found to be equivalent in effectiveness to the latter . Protein-synthesis levels in 0.3, 0.1, 0.03, and 0.01 mM valine were maximum at 0.9, 0.3, 0.09, and 0.03 mM leucine, respectively . Valine transport into the cell was found to be inversely proportional to the extracellular leucine concentration . DL-Norvaline and DL-norleucine were not effective . Valine-leucine interactions were suggested to be involved in intracellular protein synthesis.

Bone Miner, 1993 Jan, 20(1), 67 - 78
Bone-resorbing activity of different periprosthetic tissues in aseptic loosening of total hip arthroplasty; Ohlin A et al.; Using bone organ culture techniques, we studied the production of bone-resorption stimulating factors by the different tissues surrounding loosened total hip arthroplasties . Revision arthroplasties were performed in 13 patients . Various periprosthetic tissues were obtained from these patients . Culture media conditioned by the newly formed capsules contained higher bone-resorption activity when compared to the resorptive activity found in media conditioned by bone-cement membranes . Addition of indomethacin or structurally unrelated non-steroidal anti-inflammatory drugs, or corticosteroids, to bone-cement membrane cultures altered the process of bone resorption in the vicinity of cemented joint implants suggesting that both prostaglandin and other mediators may be involved in the mechanism by which bone-resorbing osteoclasts are stimulated . Our observations indicate that, in the sequence of events that leads to loosening of implants, mediators released from the joint capsules may be more important than those produced by the bone-cement membranes.

Nippon Shokakibyo Gakkai Zasshi, 1993 Jan, 90(1), 9 - 15
{Effect of prostaglandin E2 on the proliferation of cultured guinea pig gastric mucous cells}; Nakano O et al.; To understand the mechanism by which prostaglandins (PGs) preserve gastric mucosal integrity, we established a primary cultured monolayer of guinea pig gastric mucous cells, and by using this culture system, we studied whether endogenously released PGE2 could influence the proliferation of the mucous cells . By the histochemical and morphological analysis at 24h of the culture periods, the cells were recognized to contain PAS-positive mucous granules with only 3% of them being parietal cells . Although the cells which were simultaneously labeled with {3H} arachidonic acid in 0.5% serum-containing medium synthesized and released radiolabeled PGE2, PGI2 and PGA2, the release of PGE2 was more markedly observed and was partially dependent upon arachidonic acid added to the culture medium . By radioimmunoassay of the culture media, the mucous cells were found to release PGE2 in a time-dependent manner in response to 10% serum . Pretreatment of the cells with 10(-4)M indomethacin not only inhibited PGE2 release but also inhibited increase in cell number . However, the addition of PGE2 dose-dependently restored the indomethacin-induced inhibition of cell growth with the maximal increase almost to the control level at 10(-6)M PGE2 . These results suggest that PGE2 endogenously released from the cells may exert a proliferative effect on gastric mucous cells.

Ann Hematol, 1993 Jan, 66(1), 27 - 31
Effects of chemotherapeutic and immunosuppressive drugs on the production of erythropoietin in human hepatoma cultures; Wolff M et al.; The effects of agents used in combination chemotherapy were studied on erythropoietin synthesis in cultures of the human hepatoma cell line, HepG2 . Erythropoietin was measured by radioimmunoassay in the culture media after 24-h treatment periods . The RNA synthesis-inhibiting drugs daunorubicin, cyclophosphamide, ifosfamide, and cis-diamine-dichloroplatinum produced a dose-dependent decrease of the production of erythropoietin . Inhibition was also induced by the tubulin-binding agents vincristine and colchicine and the immunosuppressive agents azathioprine and cyclosporin A . The DNA synthesis-inhibiting drugs methotrexate and cytosine arabinoside, the antimycotics 5-fluorocytosine and amphotericin B, and glucocorticoids did not inhibit . Viability studies showed that the inhibition of erythropoietin production was partly correlated with cytotoxicity . These data could be relevant with respect to recombinant erythropoietin replacement therapy in tumor-associated anemia.

J Orthop Res, 1993 Jan, 11(1), 122 - 30
Differentiating and antitumor activities of 1 alpha,25-dihydroxyvitamin D3 in vitro and 1 alpha-hydroxyvitamin D3 in vivo on human osteosarcoma; Tsuchiya H et al.; The differentiating and antitumor activities of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in vitro and 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) in vivo were studied with a human osteosarcoma cell line (OST strain) . Anti-tumor activity was estimated with the use of 3-(4,5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT) assay, colony-forming assay, and athymic mouse assay . The intracellular alkaline phosphatase (ALP) activity of tumor cells and production of bone Gla protein (BGP) in culture media were measured to mark osteoblastic differentiation . In addition, the combination of 1 alpha,25(OH)2D3 and cis-dichlorodiammineplatinum(II) (CDDP) was tested by the colony-forming assay and the measurement of ALP activity and BGP production for differentiating and antitumor effects . The assays revealed that 1 alpha,25(OH)2D3 exerted a dose-related, growth-inhibitory influence . In the colony-forming assay, the 1 alpha,25(OH)2D3-treated colonies were smaller than the untreated colonies . The ALP activity and the BGP production also increased in relation to dose . In the assay in athymic mice, the relative weight of tumors treated with 1 alpha(OH)D3 at 2.5 nmol/kg was significantly smaller than that of the controls, and no side effects were observed in the 1 alpha(OH)D3-treated mice . Marked tumor chondrogenesis was observed in human osteosarcoma treated with 1 alpha(OH)D3 in athymic mice . The combination of 1 alpha,25(OH)2D3 at 10(-8) M and CDDP at 2 micrograms/ml significantly enhanced both the differentiation and the growth inhibition in vitro . Our study apparently is the first demonstration that vitamin D3 metabolites have an antitumor and differentiating effect on human osteosarcoma cells in vitro and in athymic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Dig Dis Sci, 1993 Jan, 38(1), 137 - 41
A neutrophil chemotactic factor present in H . pylori but absent in H . mustelae; Kozol R et al.; This study was designed to compare the capabilities of Helicobacter pylori and Helicobacter mustelae to generate neutrophil chemotactic activity (NCA) in vitro . H . pylori and H . mustelae were grown in parallel cultures under identical conditions . The cultures were washed and transferred to saline solution for 3 hr to avoid detecting nonspecific chemotactic activity from culture media . Supernatants were subjected to size-exclusion HPLC . All peaks from HPLC were collected and assayed for NCA . Peaks having significant NCA were subjected to gel electrophoresis . H . pylori generated 85.9 +/- 1.7% NCA compared to only 41.6 +/- 2.5% for H . mustelae (P < 0.001) . The HPLC peak containing the highest NCA from H . pylori revealed a band on gel electrophoresis at approximately 10.5 kDa . This band was not present on gels from H . mustelae . We conclude that H . pylori produces a neutrophil chemotactic factor lacking from H . mustelae . This offers an explanation for the histologic difference between gastritides caused by these organisms.

Am Rev Respir Dis, 1993 Jan, 147(1), 87 - 91
Increased granulocyte/macrophage colony-stimulating factor production by mononuclear cells from peripheral blood of patients with bronchial asthma; Nakamura Y et al.; The in vitro production of granulocyte/macrophage colony-stimulating factor (GM-CSF) by mononuclear cells (MNC) from the peripheral blood of patients with bronchial asthma (BA) was examined by enzyme-linked immunosorbent assay (ELISA) . GM-CSF concentrations in the media of MNC from patients with BA cultured with interleukin-2 (IL-2) was 80.2 +/- 52.0 pg/ml (mean +/- SE, n = 12), and that in cultures without a stimulant was 12.1 +/- 11.3 pg/ml . The GM-CSF concentrations in the media of MNC from patients with other diseases (n = 13) and from healthy volunteers (n = 6) cultured with or without IL-2 were less than 7.5 pg/ml (the minimum detectable value) . The culture media of MNC from patients with BA demonstrated activities for stimulating the proliferation and survival of eosinophils, and these activities were partially inhibited by anti-GM-CSF antibodies . GM-CSF production by MNC of patients with BA treated with glucocorticoids was lower than that of MNC from untreated patients with BA, and it was inhibited by coculture with glucocorticoids in vitro . These results suggest that GM-CSF production by MNC is increased in patients with BA, is modulated by glucocorticoids, and may play an important role in the pathogenesis of BA.

Fertil Steril, 1993 Jan, 59(1), 192 - 6
The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization; Fleetham JA et al.; OBJECTIVE: To determine whether the mouse embryo culture system can be sensitized to provide improved differentiation of suboptimal culture media for in vitro fertilization . DESIGN: Mouse embryo development in media prepared from one of three water sources were compared using embryos from two mouse strains, culturing embryos from either zygote or two-cell stage, and pretreating with either zona removal and/or cryopreservation . SETTING: Academic research department, tertiary care referral center . RESULTS: Embryos from CD1 mice were able to develop in suboptimal culture conditions, even when pretreated with zona removal or cryopreservation . Embryos from B6CBA/F1J mice were more sensitive to suboptimal culture conditions when harvested at the zygote stage than at the two-cell stage, and this sensitivity was improved after zona removal before culture . CONCLUSIONS: The mouse embryo culture system has deficiencies as an assay of culture medium quality, but the sensitivity of the assay can be optimized by harvesting at the zygote stage from an appropriate strain and by zona pellucida removal before culture.

Mol Reprod Dev, 1993 Jan, 34(1), 47 - 52
Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells; Byskov AG et al.; We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes . We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days . In this study MIS media are spent culture media from 24 hour cultures of minced adult mouse testes . In the bioassay one gonad of each fetus is cultured either in MIS medium, in control medium with forskolin, or in MIS medium with forskolin . The other gonad serves as the control and is cultured in control medium . After culture the gonads are fixed, squashed, and DNA-stained . In these preparations germ cells and somatic cells can be distinguished, and the number of germ cells in the different stages of meiosis is counted as is the number of somatic cells in mitosis . MIS activity is defined to be present in a medium when meiosis is induced in male germ cells during culture . We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner . In addition, MIS media and forskolin acted synergistically by inducing meiosis . Female germ cells seem to be unaffected by the various culture media . These findings indicate that receptors for stimuli of meiotic initiation may exist in germ cells or neighbouring somatic cells . In addition to induction of meiosis, MIS media and forskolin also dose dependently increase the number of male germ cells compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Respir Cell Mol Biol, 1993 Jan, 8(1), 28 - 34
Granulocyte/macrophage colony-stimulating factor stimulates human polymorphonuclear leukocytes to produce interleukin-8 in vitro; McCain RW et al.; Interleukin-8 (IL-8) is a potent chemotactic factor for polymorphonuclear leukocytes (PMN) . Here we examine whether PMN synthesize and release IL-8 in response to stimulation by selected inflammatory cytokines . PMN isolated from normal heparinized peripheral human blood were incubated in RPMI culture medium at 37 degrees C in 5% CO2, with and without granulocyte/macrophage colony-stimulating factor (GM-CSF) . The culture supernatants were tested for chemotactic activity using a modified Boyden chamber . Immunoreactive IL-8 protein was measured by ELISA with a monoclonal antibody specific for IL-8 . GM-CSF (0.01 to 50 ng/ml) stimulated PMN to produce chemotactic activity in a dose- and time-dependent manner . The amount of chemotactic activity reached maximal levels after 3 h of incubation with GM-CSF . Treatment of culture media supernatants with rabbit antiserum against IL-8 blocked the GM-CSF-induced chemotactic activity . IL-8 protein concentrations detected by ELISA closely paralleled the chemotactic bioactivity in both the dose-response and kinetic studies . Northern blot analysis of total RNA from PMN using a 30 mer oligonucleotide complementary to mRNA for IL-8 yielded a single 1.6-kb band . Its intensity increased 4-fold 2 h after treatment of PMN with GM-CSF . These data suggest that peripheral blood PMN can be stimulated by GM-CSF to synthesize and secrete bioactive IL-8 . Since both IL-8 and GM-CSF accumulate in sites of acute inflammation, PMN may induce IL-8 gene expression in response to GM-CSF and thereby amplify the acute inflammatory response by recruiting additional PMN into inflammatory sites.

Life Sci, 1993, 53(19), 1465 - 72
High throughput assay for inhibitors of the epidermal growth factor receptor-associated tyrosine kinase; King IC et al.; We have developed a colorimetric assay for the examination of inhibitors of epidermal growth factor (EGF) receptor-associated tyrosine kinase in intact cells . EGF receptor from cells treated with inhibitors is captured by an anti-EGF receptor antibody and the phosphotyrosine content is measured by an anti-phosphotyrosine antibody . The quantitative assay does not use radioactive substances and is configured for a high-throughput format . Since it is performed in intact cells, substances lower the phosphotyrosine content on the receptor by different mechanisms will be identified . One distinct feature of the assay is that it uses the natural substrate inside the cell as compared to others using artificial substrates in an unphysiological environment . This assay is easy to perform, is reproducible, and is compatible with many organic solvents and tissue culture media . Thus, it is useful for the discovery of EGF receptor kinase inhibitors from natural products or synthetic compounds and is particularly suitable for large-scale screening.

C R Acad Sci III, 1993, 316(4), 425 - 30
Modification of prostaglandin E2 and collagen synthesis in keratoconus fibroblasts, associated with an increase of interleukin 1 alpha receptor number; Bureau J et al.; Keratoconus, a bilateral corneal disease, is characterized by modifications in corneal shape and thinning of the stroma . It affects young people . From a biochemical point of view, a decrease in collagen content, probably due to the high collagenase activity, has been reported . In these experiments, we have studied the membrane receptors for interleukin 1 alpha, and the corresponding dissociation constant (KD) . We also investigated synthesis of prostaglandin E2 (PGE2) and collagen, as well as kinetics of cyclooxygenase . Data from normal human corneas and from keratoconus were compared . Fibroblasts from keratoconus proved to bear four fold more IL1 binding sites than those from normal cornea, with similar KD . Both types of cells synthesize prostaglandin E2, even if IL1 is not added to the medium, but the keratoconus cells produce ten times more than the normal cornea cells . When the cells are stimulated with IL1, synthesis of PGE2 strongly increases and the amounts produced by keratoconus cells are always higher than those of the normal cornea cells . Kinetics of the cyclooxygenase show that Vmax . is 10 times higher in keratoconus than in normal cornea cells; Km's are the same . The amounts of collagen synthesized by keratoconus cells are slightly lower than those of normal cornea cells . Addition of IL1 to the cultures enhances synthesis of collagenase by the cells and decreases collagen found in the culture media . The drop of collagen is more important in keratoconus than in normal cornea cell cultures.

J Dev Physiol, 1993 Jan, 19(1), 29 - 36
Prostaglandins stimulate fluid secretion in human fetal lung; McCray PB Jr et al.; Fluid secretion by the fetal pulmonary epithelium is thought to be important for normal lung development yet little is known about factors regulating its production . As prostaglandins are synthesized in human fetal lung and stimulate secretion in a variety of epithelia, we investigated the effect of prostaglandins E2 and F2a (PGE2 and PGF2 alpha) on ion transport and fluid secretion in cultured first trimester human fetal lung tissue explants . We used conventional microelectrodes to continuously record the transepithelial potential difference (psi t) . The addition of either PGE2 or PGF2 alpha to the bathing solution significantly hyperpolarized the lumen negative psi t and the subsequent addition of bumetanide, an inhibitor of chloride secretion in other systems, depolarized psi t by approximately 60% suggesting chloride transport contributed to the voltage . To assess whether this acute change in psi t represented stimulation of fluid secretion, we measured the change in luminal area of the explants after a 24-h exposure to prostaglandins . Both PGE2 and PGF2 alpha caused significant increases in the mean % luminal area of the explants compared with control tissues consistent with a stimulation of lung fluid secretion . Cultured lung tissue explants produced prostaglandins E2 and F2 alpha as assessed by radioimmunoassay of cell culture media samples and both prostaglandins stimulated cAMP accumulation in the explants . These findings show that lung fluid secretion in the human fetal pulmonary epithelium can be stimulated by prostaglandins . This effect may be mediated through cAMP dependent pathways . Prostaglandins may play a physiologic role in regulation of fetal lung fluid transport in vivo.

Acta Neuropathol (Berl), 1993, 85(2), 167 - 74
Pathological and experimental investigations in a case of gigantism; Fazekas I et al.; A pituitary adenoma was transsphenoidally removed from a 4.5-year-old girl suffering from gigantism . Prior to the operation both the growth hormone (GH) and the prolactin (PRL) levels in the serum were elevated . By light microscopy the tumor appeared to be an acidophilic adenoma . Two distinct cell types, the densely granulated and the sparsely granulated cells, could be distinguished by electron microscopy . Double immunolabeling revealed the presence of GH alone in some densely granulated cells and PRL alone in some sparsely granulated cells, as well as GH and PRL co-localized in both of the morphologically distinguished cell types . Both cell types were identified in the monolayer and the suspension cultures by electron microscopy . GH and PRL concentrations in the culture media were measured by radioimmunoassay . The basal secretion of growth hormone was almost uniform during the 3-week cell culture period . GH and PRL release was significantly inhibited by bromocriptine . Our studies revealed a bimorphous and bihormonal mixed adenoma in childhood.

Reprod Nutr Dev, 1993, 33(2), 121 - 7
Production of estradiol by the fetal rat testis; Weniger JP et al.; Testes from 17- to 20-d-old rat fetuses were cultured in vitro for various time intervals in Medium 199 alone or with added gonadotrophins . Estradiol released into the culture media was determined by radioimmunoassay . A basal estradiol secretion rate was inexistant or undetectable at all stages studied . In early stages (17 and 18 d) there was no difference in the stimulatory effect of FSH or LH at the same concentration . At 19 d, the prevalence of FSH became apparent . At 20 d, a significant action of FSH was noted after only 3 h culture time . 1-Methyl-1, 4-androstadiene-3,17-dione, an aromatase inhibitor, markedly depressed FSH-stimulated estradiol secretion . In the 20-d old testis, stimulation of estradiol production by FSH was more rapid and reached a higher level than by (Bu)2cAMP . It is suggested that the difference in the action of LH and FSH reflects the difference in the time of appearance of the corresponding receptors in the developing fetal testis.

Folia Biol (Praha), 1993, 39(1), 29 - 39
Inhibition of growth of the lymphocyte lines by deferoxamine under various iron-supply conditions; Kovar J et al.; Inhibition of growth of the lymphocyte lines by iron-binding agent deferoxamine under various iron-supply conditions was studied . Three different defined culture media, representing three different iron-supply conditions, were used: (1) ferric citrate (500 microM) medium, (2) transferrin (5 micrograms/ml) medium, and (3) low-iron medium (this medium without any iron compound added contains 0.6 microM contaminative non-transferrin iron) . Mouse B cell line PLV-01, human T cell line Jurkat, and human B cell lines Raji and HSCE- were employed . Raji and HSCE- cells are able to grow in low-iron medium but PLV-01 and Jurkat cells do not grow in the medium . For all cell lines tested in ferric citrate medium, a 50% growth inhibition was achieved with 440-500 microM deferoxamine . In transferrin medium, deferoxamine concentrations of 4.4-5.5 microM were required for a 50% inhibition of PLV-01, Jurkat and HSCE- cells . For the same degree of inhibition of Raji cells, 39 microM deferoxamine was required . In the case of low-iron medium, a 50% inhibition of Raji and HSCE- cells was achieved with about 1.4-2.1 microM deferoxamine . The data demonstrate that the sensitivity of the lymphocyte lines to deferoxamine depends on iron-supply conditions . Under the same iron-supply conditions, individual cell lines can exhibit different sensitivity . However, the sensitivity does not correlate with the ability of the cell lines to grow in low-iron medium.

Bioelectromagnetics, 1993, 14(3), 187 - 96
Effect of static magnetic fields on osteoblasts and fibroblasts in vitro; McDonald F; In vitro assays were made of the effect of a static magnetic field of a neodymium magnet on cellular behavior . The cell turnover rate was examined by the incorporation of radioactive thymidine, and anabolic processes were measured by the incorporation of radioactive proline . Cell cultures of fibroblast- and osteoblast-like cells of the neonatal rat calvarium were assayed to determine uptakes of radioactive thymidine and proline; these assays were performed in conjunction with examination of an explant of the rat calvarium . The cells were assayed after exposure to a field for 1-, 3-, 5-, 7-, and 10-day periods . Cells were exposed to north and south poles with a pole-face flux density of 0.61 T; control cultures were exposed to an unmagnetised piece of neodymium . After sham exposure or exposure to the magnetic field, 50 microCuries/ml of culture media of isotope were added to the culture medium . The cultures were returned to an incubator for 6 h . Then, following centrifugation, the supernatant was assayed for radioactivity in a scintillation counter after addition of 3 ml of scintillation fluid . A statistically significant magnetic stimulation of turnover rate and synthesis of fibroblasts was found, but stimulation of osteoblasts did not occur . Conversely, the explants, which represent the osteoblasts and fibroblasts in an organised system, showed a statistically significant inhibition in uptake of the radioactive label . The data indicate both variability and diversity of cellular behaviour, and they accentuate the need for caution in the interpretation of effects of static magnetic fields.

Exp Clin Endocrinol, 1993, 101(4), 255 - 61
Different steroidogenic response of young and aged porcine small and large luteal cells to prostaglandin F2 alpha, oxytocin and estradiol; Pitzel L et al.; The role of oxytocin (OXT) and prostaglandin F2 alpha (PGF2 alpha) in the process of luteal regulation, particularly their function in the early luteal phase is poorly understood . Therefore the effects of both compounds on in vitro steroid release of porcine luteal cells harvested from young/middle-aged (day 4-6, day 0 = 1st estrous day) or old (day 12-14) corpora lutea were tested . As corpora lutea (CL) contain at least two different steroidogenic cell populations, fractions of the so called small (SLC) and large (LLC) luteal cells were prepared and tested in separate experiments . In SLC as well as LLC from young CL OXT and PGF2 alpha inhibited progesterone (P) production but induced a strong increase of estradiol (E2) release . In old SLC and LLC OXT and PGF2 alpha were still inhibitory to P release but OXT was ineffective and PGF2 alpha had a moderate stimulatory effect on luteal E2 secretion . In SLC cultures from young but not from old CL E2 exerted a powerful stimulatory effect on progesterone (P) secretion, i.e . E2 has strong luteotrophic effects in the early luteal phase . Indeed, the pronounced inhibitory effect of OXT and PGF2 alpha on P release from SLC could be counteracted by the addition of exogenous E2 to the culture media . Therefore, we suggest that in the early luteal phase OXT as well as PGF2 alpha have an indirect, E2-mediated luteotrophic effect on P release which is stronger than the direct inhibitory action on P secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Chem Toxicol, 1993, 16(4), 321 - 39
Culture medium composition affects the relative toxicities of chlorobenzenes in rat liver slices and the isolated perfused liver; Fisher RL et al.; The effects of different media composition on the hepatotoxicity produced by monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB) and 1,4-dichlorobenzene (1,4-DCB) were examined in two different in vitro systems . The toxicity of these chlorobenzenes was investigated in the perfused rat liver and liver slices using Krebs-Henseleit buffer . Significant differences between the chlorobenzenes were apparent in the perfused liver but not in the tissue slices . However, a dose and time related response of rat liver slices to the chlorobenzenes was observed . Partial amelioration of the chlorobenzene toxicity was observed when the Krebs-Henseleit buffer was supplemented with vitamins, amino acids, and/or bovine serum albumin . 1,2-DCB and 1,3-DCB toxicity was affected by amino acids and vitamins . The toxicity produced by 1,4-DCB was suppressed by amino acids, vitamins and 1% BSA . MCB hepatoxicity could only be suppressed by 1% BSA . This data suggests that tissue culture media composition plays a major role in the hepatotoxicity of the chlorobenzenes.

Jpn J Physiol, 1993, 43 Suppl 1, S193 - 6
Response of aged neurons to super-hypotonic environment; Horie H et al.; Cultured sensory neurons from aged mice (24 months old) lost capability of cell volume auto-regulatory mechanism after the rapid treatment with a hypotonic solution of 1/4 the normal osmolarity or lower . However, they could survive and extend neurites in super-hypotonic media such as 1/4 osmolar and even 1/8 osmolar culture media when the osmolarity was reduced gradually to such a low level.

Biol Cell, 1993, 78(3), 191 - 7
Triiodothyronine influences quail myoblast proliferation and differentiation; Marchal S et al.; The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2,500 and 7,000 cells/cm2 . Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts) . Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and 3H-thymidine incorporation . However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation . Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation . In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts . This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot . All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle . The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.

J Reprod Fertil Suppl, 1993, 47, 189 - 201
In vitro fertilization in domestic and non-domestic cats including sequences of early nuclear events, development in vitro, cryopreservation and successful intra- and interspecies embryo transfer; Pope CE et al.; The domestic cat may be used as a model for developing assisted reproduction techniques including in vitro fertilization (IVF), embryo culture, cryopreservation and embryo transfer (ET) for application to threatened and endangered species of non-domestic cats . Interoestrous domestic cats were injected with a total of 1.0-6.0 mg follicle-stimulating hormone (FSH) daily for 4 days and with 100 iu human chorionic gonadotrophin (hCG) on day 5 . Follicular oocytes recovered at 26 +/- 1 h after hCG were co-incubated for 4-6 h at 38 degrees C in 5% CO2 with spermatozoa (1-2 x 10(5) motile spermatozoa ml-1) collected by artificial vagina . To determine the timing of sperm penetration and early fertilization events in vitro, oocytes were fixed and examined at intervals from 0.5 to 10 h after sperm exposure . The penetration rate of metaphase II (MII) oocytes at 0.5-3 h was equivalent to that at 3-6 h (95 versus 96%) . Second polar body extrusion, pronuclear formation and apposition were observed at 2, 6-8 and 10 h, respectively . Simple (Tyrode's) and complex (F-10, M-199 and CMRL-1066) culture media with 10% fetal calf serum were compared for their ability to support development to the morula or blastocyst stage during culture periods of 96-168 h after IVF . The average number of cells per embryo was similar (P > 0.05) in the various media at each interval except that CMRL-1066 reduced (P < 0.05) development at 96 h if it was used before the two-cell stage . In F-10, neither the presence of intact cumulus cells nor changing to fresh F-10 medium at 48 h affected development at 96 h . Blastocyst development at 168 h was similar in both F-10 (18%) and Tyrode's (26%) . To determine developmental ability in vivo, IVF-derived embryos (n = 586) were transferred at 96 or 120 h to recipients (n = 49) that had undergone synchronous oocyte recovery as donors . The percentage of recipients producing kittens after transfer of embryos cultured for 96 or 120 h in F-10 was 31 and 25, respectively, compared with 55% of 120 h recipients receiving embryos cultured in M-199 or Tyrode's . Overall, more pregnancies occurred following transfer of > or = 12 embryos (11/26) than if < 12 embryos were transferred (6/23).(ABSTRACT TRUNCATED AT 400 WORDS)

Connect Tissue Res, 1993, 29(3), 223 - 30
Direct extraction of gelatinases from rat bone; Bollen AM et al.; Many studies have shown that gelatinases are secreted into the medium of cultures of various cell and tissue types, including bone cells . It is not clear, however, to what extent the culture process is responsible for inducing the expression of these proteases . In the present study, gelatinolytic enzymes were extracted directly from bone and other tissues and identified as bands of activity on SDS-PAGE enzymograms using gelatin as the substrate . Two forms of gelatinase (72-kDa and 92-kDa) were present in extracts of normal young rat bone . Yields were markedly higher from compact bone than from other tissues (blood, marrow, tendon, cancellous bone, articular cartilage, and skin) . More 92-kDa than 72-kDa gelatinase was extracted from bone . The proteolytic specificity of the 92-kDa gelatinase isolated from the bone extract was shown to be similar to that reported for the enzyme isolated from tissue culture media . Native type I collagen was not cleaved but heat denatured type I collagen (gelatin) and native type IV, type V, type IX and type XI collagens were degraded . The proteolytic activity was inhibited by EDTA . The results indicate that more gelatinases can be extracted from bone tissue than from other tissues using mild extraction conditions . The cellular origin and function of these enzymes in bone remain to be defined.

Pathobiology, 1993, 61(2), 98 - 103
Type I collagen synthesis in cultured aortic smooth muscle cells from spontaneously hypertensive rats and normotensive control, Wistar-Kyoto rats; Akita M et al.; An inhibition ELISA was used to quantify the amount of type I collagen synthesized in culture media and cell layers from aortic smooth muscle cells (SMCs) of spontaneously hypertensive rats (SHR) and normotensive control, Wistar-Kyoto rats (WKY) . Cultured cells were also observed by electron microscopy . Collagen content in the culture media was strongly increased after 6 days in both cultures . Collagen and protein contents in the medium and cell layer from SHR were significantly higher than those in WKY at day 14 . However, cell density in SHR-derived cells was also higher than that of WKY . No significant differences were detected in the rates of collagen content between SHR and WKY on a per cell basis . The main differences between SHR and WKY in collagen and protein levels may be due to the greater number of SHR cells and increased amounts of extracellular matrix components . The assay system outlined here should be useful for studying the control of extracellular-matrix synthesis.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1993, 11(2), 89 - 92
{Role of macrophage in presenting antigens of Schistosoma japonicum}; Fang XH et al.; Indirect ELISA was employed to monitor the serum anti-UEA (urea soluble egg antigen of Schistosoma japonicum) antibody level of mice immunized by: a . UEA pulsed macrophage (Mphi+); b . Cultural supernatant of Mphi+; c . paraformaldehyde fixed M phi (P-Mphi) pulsed with UEA; d . Ammonium chloride treated M phi (NH4Cl-Mphi) pulsed with UEA; e . P-Mphi pulsed with trypsin digested UEA (T-UEA); f . NH4Cl-Mphi pulsed with T-UEA . The normal Mphi, its supernatant and the culture media RPMI 1640 acted as the negative control . The results showed: 1 . Serum anti-UEA antibody levels of mice immunized by a and b raised markedly, indicating that the immunogenicity of UEA might be kept up after Mphi processing and the antigenic message could be transferred either by the Mphi+ or by its supernatant; 2 . Mice immunized by c and d gave similar results, but the anti-UEA antibody level of the former was higher than that of the latter, suggesting that polyformaldehyde could not alter the UEA binding site on the surface of Mphi; 3 . In the case of mice immunized by e and f, the antibody levels were much lower than that of mice immunized by c and d, suggesting that UEA binding sites on Mphi surface as well as UEA immunogenicity could be changed by trypsin.

Nat Toxins, 1993, 1(5), 303 - 7
Rapid colorimetric bioassay for screening of Fusarium mycotoxins; Rotter BA et al.; The cytotoxicity of Fusarium mycotoxins was evaluated using a trichothecene sensitive cell line (BHK-21, baby hamster kidney cells) in combination with the MTT-cleavage test as an end-point measurement . Cells tended to be more sensitive to the type A trichothecenes with midpoint cytotoxicity values ranging from 1.6 ng/ml for T-2 toxin to 60 ng/ml for scirpentriol . The cytotoxicity value for deoxynivalenol (type B) was 112 ng/ml . The inherent disadvantage of the MTT-assay (formation of insoluble formazan) was overcome by using the analog MTS and measuring the water-soluble formazan directly in the culture media . The MTS-midpoint cytotoxicity values for T-2 toxin and deoxynivalenol (2.1 and 141 ng/ml, respectively), although slightly higher, showed a good correspondence to the MTT-test . Both the MTT- and MTS-cleavage tests are useful for evaluating the cytotoxicity of Fusarium mycotoxins . The replacement of MTT by MTS substantially reduced the number of sample processing steps and the length of time required to complete the cytotoxicity assay.

Reprod Fertil Dev, 1993, 5(4), 445 - 58
Embryonic development in culture of the marsupials Antechinus stuartii (Macleay) and Sminthopsis macroura (Spencer) during preimplantation stages; Yousef A et al.; Forty-nine blastocysts from 11 brown antechinus, Antechinus stuartii, and 96 blastocysts from 17 stripe-faced dunnarts, Sminthopsis macroura, were used to develop a culture system for embryos during preimplantation stages . Blastocysts of brown antechinus were collected on Days 6-9 for unilaminar stages, Days 16-21 for bilaminar stages and Days 20 and 21 for trilaminar stages . Blastocysts of stripe-faced dunnarts were collected on Day 6 for unilaminar stages, Days 6-8 for bilaminar stages and Day 8 for trilaminar stages . Culture media were Dulbecco's modified Eagle's medium (DMEM) with 4.5% glucose and Whittingham's T6 medium both of which were supplemented with 5, 10, 12.5 and 20% fetal calf serum (FCS) . Antechinus serum (5%) and bovine serum albumin (0.1%, 0.2%) were also added to some media . Human amniotic fluid (HAF) and Monomed media were also tested . Blastocysts were cultured at 35 degrees C in 5% CO2 in air . DMEM + 10% FCS and HAF supported normal development for the longest periods and over the greatest range of stages . Developmental failure of blastocysts in vitro during expansion of the unilaminar blastocyst and formation of the bilaminar blastocyst suggests that these stages may be dependent on uterine signals . When cultured in DMEM + 10% FCS, the rate of development of bilaminar and trilaminar blastocysts into organogenesis was 4 h slower than in vivo in the stripe-faced dunnart and about 6 h slower than in vivo in the brown antechinus . Embryos of stripe-faced dunnarts were cultured to within 18 h of birth.

J Cell Sci Suppl, 1993, 17, 41 - 7
An electron microscopic study of TGN38/41 dynamics; Ladinsky MS et al.; We have used electron microscopy to further characterize details of the dynamics of TGN38/41, a protein found to cycle between the trans-Golgi network and the plasma membrane . Immunogold-labeling of NRK cells under steady-state conditions shows the majority of TGN38/41 is localized to the trans-most Golgi cisternae and the trans-Golgi network . Small amounts of this molecule can be detected in early endosomes . Capture of cycling TGN38/41 molecules at the cell surface altered the steady state distribution . This was accomplished by binding TGN38/41 luminal domain antibodies to solid supports (beads), which were introduced to the culture media of cells . As increasing numbers of antigen-antibody complexes formed, the beads were internalized by the 'zippering mechanism' of phagocytosis . This provides a system that can address many questions related to the function of TGN38/41 and the trans-Golgi network itself.

Invest Clin, 1993, 34(2), 85 - 98
{Gum-like exudate from Laguncularia racemosa (white mangrove) as culture media for fungi}; Mesa LM et al.; Morphological studies of eight species of fungus: Aspergillus flavus Microsporum canis, Epidermophyton floccosum, Curvularia lunata, Cladosporium carrionii, Natrassia mangifera (Edo . Scytalidium), Sporotrix schenckii y Rhizophus oligosporus, which belong to families Mucedinaceae, Dematiaceae and Mucoraceae have been carried out in support medium based in gum exudate from Laguncularia racemosa (mangle blanco) . This native polimer contains galactose, arabinose, rhamnose, uronic acid and proteins . Nitrogen calcium and magnesium are microconstituents of the gum . An economical substrate which contained gum exudate (4%) and agar (1.5%) was used in these studies . The results obtained showed that gum exudate-agar medium (EGA) permits an adequate identification of the studied species, therefore, it is a possible substitute for Sabouraud . It is important to know that the gum exudate is a natural product, economical and easy to obtain.

Neurotoxicology, 1993 Spring, 14(1), 35 - 40
N-methyl-D-aspartate receptors mediate cyanide-induced cytotoxicity in hippocampal cultures; Patel MN et al.; We reported previously that glutamate excitotoxicity may contribute to cyanide-induced neuronal injury . Cyanide stimulates glutamate release which can activate glutamate receptors to initiate excitotoxic processes . This study examines the role of EAA receptor subtypes in mediating cyanide-induced cytotoxicity . Cytotoxicity was assessed in primary rat hippocampal cultures by measuring lactate dehydrogenase (LDH) in the culture media . NaCN (0.1-10 mM) or glutamate (0.01-1 mM) produced concentration-dependent cytotoxicity following 18 hrs of incubation . Glutamate-induced cytotoxicity was partially blocked by the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA antagonist, 2-amino-5-phosphonovalerate (APV) . Simultaneous exposure of cultures to both CNQX and APV provided complete protection against glutamate cytotoxicity . NaCN-induced cytotoxicity was not blocked by CNQX, but completely blocked by APV and simultaneous exposure to CNQX and APV did not offer added protection . These results indicate that in hippocampal cultures, both non-NMDA and NMDA receptors mediate glutamate excitotoxicity, whereas NaCN-induced cytotoxicity is mediated primarily by activation of the NMDA receptors.

Zh Mikrobiol Epidemiol Immunobiol, 1993 Jan-Feb, (1), 3 - 7
{The production of a protein base for nutrient media from a dairy industry byproduct}; Rodriguez Martinez S et al.; The technology of obtaining protein hydrolysate, a by-product of butter making, has been developed . This hydrolysate has been successfully tested as protein base for the preparation of culture media used in the quality control of foodstuffs and the diagnosis of infectious diseases.

Cytotechnology, 1993, 13(3), 161 - 73
Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media; Mochizuki K et al.; It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium . Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose . It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture . These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium . An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production and vice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.

Cytotechnology, 1993, 11(3), 213 - 8
Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich; Suzuki M et al.; Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days . It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion . These facts suggest that the membrane-supported collagen sandwich mimics well the in vivo environment of hepatocytes . This method has great potential for the long-term culture of primary cells.

Neoplasma, 1993, 40(3), 141 - 6
Phorbol ester-induced modulation of cell surface antigens on U-937 cells in protein-free medium: effect of protein kinase and calmodulin inhibitors; Hunakova L et al.; Phorbol ester (PMA) induced decrease in cell surface expression of CD4 antigen was potentiated on human monoblastoid cell line U-937 cultured and induced in protein-free culture medium . Down-regulation of the CD4 antigen was partially inhibited by the isoquinoline protein kinase inhibitor H7 and unaffected by the calmodulin inhibitor R24571 (calmidazolium) . PMA-induced increase of the monocyte-associated CD14 antigen was dependent the presence of fetal bovine serum (FBS) in culture medium and partially abolished by calmodulin inhibitor R24571 . H7 inhibitor partially abrogated this up-regulation in 4 out of 8 induction experiments . PMA-induced down-regulation of MHC class I antigen was found in both FBS-supplemented and protein-free culture media . This effect was partially inhibited by both H7 and R24571 inhibitors.

Prostate, 1993, 22(2), 109 - 18
Growth of an androgen-sensitive human prostate cancer cell line, LNCaP, in nude mice; Lim DJ et al.; This study was undertaken to establish an androgen-sensitive model of human prostatic carcinoma in nude mice . The androgen-sensitive prostatic carcinoma cell line, LNCaP, was suspended in reconstituted basement membrane (Matrigel) and injected subcutaneously into nude mice . The LNCaP cell line was chosen for the study, because the cell line is androgen-sensitive and secretes prostate specific antigen (PSA) into culture media . Following injection of 1 x 10(6) LNCaP cells with 0.25 ml of Matrigel, 88% of mice exhibited palpable tumor burdens after 12 weeks of observation . In addition, significant levels of PSA were observed in the serum of LNCaP-bearing mice . Bilateral orchiectomy of mice resulted in tumor regression and stabilization of serum PSA levels, compared to testis-intact controls . A significant correlation of PSA to tumor volume and weight was observed . The castrate level of testosterone was confirmed by radioimmunoassay and was similar to testosterone levels in female nude mice . Matrigel allows for a conducive environment to propagate LNCaP cells in nude mice . Furthermore, the growth can be manipulated by castration, leading to involution of tumor and stabilization of serum PSA level . This in vivo model of hormone-sensitive human prostate cancer cell line will serve as a model for the study of prostate tumor biology and treatment.

J Cardiovasc Pharmacol, 1993, 22 Suppl 8, S214 - 8
Endothelin-1 binding sites and immunoreactivity in the cultured human placental trophoblast: evidence for an autocrine and paracrine role for endothelin-1; Ferre F et al.; In human placenta, endothelin (ET) could derive from maternal, fetal, and/or endogenous sources . Therefore, localization of ET-1 was investigated by immunohistochemistry in human term placenta and in cultured trophoblasts . In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblasts of the villi . ET-1 IR was also detected in the decidual cells and in the extravillous cytotrophoblasts of the basal plate . The extravillous cytotrophoblasts of the chorionic plate and of the placental septa also exhibited strong ET-1 IR . For trophoblast culture, cytotrophoblastic cells were obtained from placental villi by trypsin-DNAse dispersion, further purified on a Percoll gradient, and enriched by employing a monoclonal anti-HLA class I antibody . After different periods of culture of purified cytotrophoblastic cells (1-5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblasts . The presence of ET-1,2 immunoreactivity (ET-1,2 IR) in the culture media was demonstrated by radioimmunoassay . A uniform daily production of the peptide was observed over at least 5 days (approximately 50 fmol/10(6) cells/24 h) . Furthermore, trophoblastic cells that had been cultured for 5 days contained significant amounts of ET-1,2 IR (24 fmol/10(6) cells) . These results suggest a trophoblastic origin for ET-1 and support the hypothesis of a paracrine and autocrine action of the peptide in the physiology of the trophoblast and placenta.

J Surg Res, 1992 Dec, 53(6), 602 - 9
Regulatory effects of interleukin-4 on tumor-infiltrating lymphocytes derived from human renal cell carcinoma; Peyret C et al.; We have studied the effects of interleukin-4 (IL-4) on the expansion, proliferation, phenotype, and antitumor activity of tumor-infiltrating lymphocytes (TIL) derived from human renal cell carcinoma . Cultures were obtained from three primary renal tumors and one group of tumor-invaded, regional lymph nodes . IL-4 induced a significant increase in lymphocyte expansion and proliferation, but the response was dependent on the concurrent dose of IL-2 in culture . Increased growth activity was only observed in those cultures receiving low doses (20 U/ml) of IL-2 (average increase of fold expansion of 6.5, P < 0.01) with no changes in growth activity in the high dose (1000 U/ml) cultures . The combination of low dose IL-2 and IL-4 (200 U/ml) promoted lymphocyte growth significantly better than high dose IL-2 alone, the current standard growth regimen for in vitro expansion of TIL . TIL grown in the presence of IL-4 significantly reduced the level of non-specific, non-major histocompatibility complex-restricted antitumor activity (P < 0.01 for allogeneic renal, nonrenal, and NK-sensitive K562 cells), while exhibiting no effect on the level of autologous killing . This is in contrast to previous findings of significant enhancement of autologous antitumor activity using IL-4 on tumor-specific, melanoma-derived TIL . We also evaluated the effects of irradiated autologous tumor stimulation (TIL:tumor ratio, 10:1) on TIL cultures . Addition of autologous tumor cells increased expansion and proliferation of all cultures regardless of concurrent lymphokines present in the culture media (average increase fold expansion of 2.21, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Biol Reprod, 1992 Dec, 47(6), 917 - 24
Estrogen- and progesterone-dependent secretory changes in the uterus of the sheep; Murray MK et al.; This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep . Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP) . Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels . The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05) . Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment . An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals . An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment . These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.

Pigment Cell Res, 1992 Dec, 5(6), 379 - 86
The effect of oxygen on melanin precursors released from retinal pigment epithelial cells in vitro; Akeo K et al.; The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells . The damage is specific to cell type and to the ambient oxygen concentration . To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells . Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations . However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed . The data provided evidence that RPE modulated dopa metabolism . Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range . The spectrum between the dopa-derived compounds in the media has an absorbance at 240-275 nm and a maximum around 300 nm with a shoulder near 375 nm . Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.

Biochem J, 1992 Dec 1, 288 ( Pt 2), 451 - 6
A non-glycosylated extracellular superoxide dismutase variant; Edlund A et al.; The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme . The importance of the carbohydrate moiety for the properties of the enzyme is unknown . An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln . The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones . The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography . The tetrameric state was retained . The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased . The enzymic activity was essentially retained . The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility . Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin . The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature . It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.

J Clin Microbiol, 1992 Dec, 30(12), 3065 - 9
Production of monoclonal antibody to a phenolic glycolipid of Mycobacterium tuberculosis and its use in detection of the antigen in clinical isolates; Cho SN et al.; A monoclonal antibody (MAbIII604) specific to phenolic glycolipid Tb (PGL-Tb), a Mycobacterium tuberculosis-specific antigen, was produced and used in the detection of the antigen . MAbIII604 reacted with the PGL-Tb antigen but not with other phenolic glycolipids from Mycobacterium leprae, M . bovis, and M . kansasii, thus indicating the specificity of the monoclonal antibody to PGL-Tb . A dot enzyme-linked immunosorbent assay with MAbIII604 was employed to detect the PGL-Tb antigen in lipids purified from M . tuberculosis clinical isolates . Of 50 isolates, 32 (64.0%) showed clear evidence of the PGL-Tb antigen by the dot enzyme-linked immunosorbent assay, but there were marked variations in the intensities and sizes of spots . This suggests differences in PGL-Tb antigen production among M . tuberculosis strains even when they are grown in the same culture media and conditions . This was most evident from the fact that in only eight (16.0%) of the isolates examined was the PGL-Tb antigen detectable by thin-layer chromatography, which is much less sensitive for the detection of glycolipid antigens . This study shows that monoclonal antibodies specific to PGL-Tb are useful in detecting the antigen in lipid extracts and that there is a marked variation in the PGL-Tb production among M . tuberculosis clinical isolates.

J Virol, 1992 Dec, 66(12), 6953 - 9
Sulfation of the human immunodeficiency virus envelope glycoprotein; Bernstein HB et al.; Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins . In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell . We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42- . Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene . N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein . The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates . Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins . Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42- . However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins . Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems . Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.

Blood, 1992 Dec 1, 80(11), 2746 - 54
Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells; Underhill DM et al.; Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells . After complexes are internalized, the t-PA component is degraded . However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated . To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C . 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments . After ligand uptake, intracellular vesicles were separated on density gradients . Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes . After 20 minutes of uptake, the complex began to appear in lysosomes . Subsequently, low molecular weight labeled ligand fragments were detected in culture media . A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation . When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed . Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation . After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes . Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.

Biochem Cell Biol, 1992 Dec, 70(12), 1319 - 24
Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells; Zha X et al.; The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated . The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine . The uptake of labelled choline was noncompetitively inhibited by amino acids . Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine . Analyses of the labelings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids . The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner . Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature . Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labelings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine . Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline . Based on the specific radioactivity of CDP-choline and the labeling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells . In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells . We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.

Int J Dev Biol, 1992 Dec, 36(4), 517 - 26
Effects of retinoids on tooth morphogenesis and cytodifferentiations, in vitro; Mark MP et al.; The first embryonic lower mouse molar was used as a model system to investigate the effects of two retinoids, retinoic acid (RA) and a synthetic analogue, Ch55, on morphogenesis and cytodifferentiations in vitro . Exogenous retinoids were indispensable for morphogenesis of bud, cap and bell-stage molars in serum-free, chemically-defined, culture media . Transferrin and RA or transferrin and Ch55 acted synergistically in promoting morphogenesis from bud and cap-stage explants . Transferrin, per se, had no morphogenetic effect . Epithelial histogenesis, odontoblast functional differentiation and ameloblast polarization always occurred in RA-depleted explants . Comparison of the distributions of bromodeoxyuridine (BrdU) incorporation between explants cultured in the absence or presence of RA revealed that RA could modify the patterns of cell proliferation in the inner dental epithelium and dental mesenchyme . Inner dental epithelium cell proliferation is regulated by the dental mesenchyme through basement membrane-mediated interactions, and tooth morphogenesis is controlled by the dental mesenchyme . Laminin is a target molecule of retinoid action . Using a monospecific antibody, we immunolocalized laminin and/or structurally-related molecules sharing the laminin B chain in the embryonic dental mesenchyme and in the dental basement membrane and showed that RA could promote the synthesis or secretion of these molecules . Based on previous in situ hybridization data, it was speculated that CRABPs might regulate the effects of RA on embryonic dental cell proliferation . The fact that Ch55, a retinoid which does not bind to CRABPs, is 100 times more potent than RA in promoting tooth morphogenesis in vitro seems to rule out this hypothesis . On the other hand, the stage-specific inhibition of tooth morphogenesis by excess RA is consistent with the hypothesis that CRABPs might protect embryonic tissues against potentially teratogenic concentrations of free retinoids.

Arerugi, 1992 Dec, 41(12), 1664 - 71
{Increased granulocyte/macrophage colony stimulating factor production by mononuclear cells from patients with bronchial asthma}; Kawaji K et al.; The in vitro production of granulocyte/macrophage colony stimulating factor (GM-CSF) by mononuclear cells from the peripheral blood of patients with bronchial asthma (BA) was examined by enzyme-linked immunosorbent assay (ELISA) . In 3 of 12 cases studied, mononuclear cells from BA patients produced GM-CSF without stimulation . And in 5 of 12 cases studied, mononuclear cells from BA patients produced GM-CSF in response to IL-2 . Mononuclear cells from patients with other diseases (n = 13) and healthy volunteers (n = 6) did not release any detectable (> or = 7.5 pg/ml) GM-CSF . The culture media of mononuclear cells from BA patients showed activities for stimulating the proliferation and survival of eosinophils, and these activities were partially inhibited by anti-GM-CSF antibodies . GM-CSF production by mononuclear cells from BA patients treated with prednisolone was lower than that of mononuclear cells from untreated BA patients . And prednisolone showed a reduction in the GM-CSF production from mononuclear cells in response to IL-2 . These results suggest that GM-CSF production by mononuclear cells may play a role in the pathogenesis of BA.

Matrix, 1992 Dec, 12(6), 481 - 7
Dissociation of collagenase-tissue inhibitor of metalloproteinases-1 (TIMP-1) complex--its application for the independent measurements of TIMP-1 and collagenase activity in crude culture media and body fluids; Yamashita K et al.; Collagenase-tissue inhibitor of metalloproteinases-1 (TIMP-1) complex was prepared from activated collagenase and TIMP-1 purified from culture media of human skin fibroblasts . After having been confirmed to be a complex by zinc chelate chromatography, the complex was demonstrated to dissociate by passage through an anti-TIMP-1 monoclonal antibody-affinity column . On the basis of above evidence, a simple strategy was set up for the independent measurements of TIMP-1 concentration, and both active and total collagenase activities in crude culture media and body fluids.

Fertil Steril, 1992 Dec, 58(6), 1250 - 3
The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos; Vasuthevan S et al.; Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos . However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO . Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.

Virology, 1992 Dec, 191(2), 973 - 7
CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins; Miller SB et al.; The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection . Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions . To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation . Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2) . Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions . We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation . Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation . However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation . The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.

Biochem Biophys Res Commun, 1992 Nov 30, 189(1), 8 - 13
Stimulation of serum-free cell proliferation by coenzyme Q; Sun IL et al.; Coenzyme Q added to culture media stimulates the growth of HeLa and Balb/3T3 cells in serum free conditions . The stimulation by coenzyme Q is additive to the stimulation by ferricyanide, an impermeable electron acceptor for the transplasma membrane electron transport . alpha Tocopherylquinone can also stimulate cell growth, but vitamin K1 is inactive or inhibitory . The response to coenzyme Q and ferricyanide is enhanced with insulin . A contribution to plasma membrane NADH oxidation or modification of the membrane quinone redox balance can be a basis for the growth stimulation.

Thromb Haemost, 1992 Nov 10, 68(5), 539 - 44
The influence of glycosylation on the catalytic and fibrinolytic properties of pro-urokinase; Lenich C et al.; We previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media . To determine whether this difference is related to the lack of glycosylation of the E . coli product, we compared the activity of E . coli-derived pro-UK {(-)pro-UK} with that of a glycosylated pro-UK {(+)pro-UK} and of a mutant of pro-UK missing the glycosylation site at Asn-302 {(-)(302)pro-UK} . The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell . The nonglycosylated pro-UKs were activated by plasmin more efficiently (approximately 2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK . Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK . These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E . coli-derived pro-UK.

J Biol Chem, 1992 Nov 5, 267(31), 22035 - 42
Down-regulation of interleukin 6 receptors of mouse myelomonocytic leukemic cells by leukemia inhibitory factor; Yamaguchi M et al.; We examined the effect of leukemia inhibitory factor (LIF) on the expression of interleukin 6 receptors (IL-6R) on mouse myelomonocytic leukemic M1 cells . Binding studies using 125I-labeled human and murine IL-6 revealed that LIF caused a decrease in IL-6 binding to M1 cells . The decrease became evident within 1 h, and the maximum decrease was observed at 3-6 h . Scatchard plot analysis revealed that M1 cells had a single class of high affinity receptors for IL-6 and that LIF-induced decrease in IL-6 binding was due to a decrease in the number of IL-6R on the cell surface and not to changes in their affinity . The affinity of IL-6R on M1 cells to human IL-6 (Kd = 2.25 nM) was about 10-fold lower than that to murine IL-6 (Kd = 200 pM) . The amount of IL-6 secreted into culture media by M1 cells that were treated with LIF for up to 12 h was not enough to cause receptor down-regulation . Northern blot analysis demonstrated that IL-6R mRNA was down-regulated by LIF treatment, and similar regulation was also observed when the cells were treated with IL-6 . The time course of the IL-6R mRNA level was similar to that of IL-6R expression on the cell surface, suggesting that the main mechanism responsible for the loss of high affinity IL-6R was the regulation of IL-6R mRNA . Although the half-life of IL-6R on the cell surface was about 30 min, the addition of LIF reduced it to 16 min, suggesting the existence of an additional mechanism responsible for the loss of high affinity IL-6R on the cell surface.

Microbiologia, 1992 Nov, 8(2), 106 - 14
Comparative study of some factors affecting enumeration of moulds using dilution plate techniques; Bragulat MR et al.; The influence of dilution plating technique, nature of diluent, culture media and incubation period on the enumeration of moulds have been studied . Three new culture media containing Auramine, Gentian Violet and Malachite Green respectively have been induced in this study . No significant differences were observed between results obtained after 3, 5 and 7 days of incubation . Significantly higher recoveries were obtained using the surface-spread method than pour plate method . Using the first technique no effect of diluent was observed, and among the different culture media studied higher counts were obtained with medium containing Auramine.

Neuroendocrinology, 1992 Nov, 56(5), 660 - 5
Enzyme-linked immunosorbent assay for rat and human luteinizing hormone; Leiva LA et al.; A sensitive and specific enzyme-linked immunosorbent assay (ELISA) suitable for measuring rat and human luteinizing hormone (LH) is described . The LH-ELISA used anti-bLH beta subunit monoclonal antibody-coated plates, an antiserum against rLH or hLH and an antibody against rabbit IgG labelled with peroxidase . Using rLH-RP-3 or hLH WHO IRP 68/40 as standard diluted in assay buffer, the LH-ELISA had a sensitivity of 50 pg/ml and 0.78 mIU/ml, respectively . The LH-ELISA allowed accurate determination of LH in buffer, cell culture media, anterior pituitary extracts and sera and was highly specific for rat and human LH . The use of this ELISA offers improvements in convenience, economy, sensitivity and safety over comparable radioimmunoassay procedures.

Biol Reprod, 1992 Nov, 47(5), 889 - 902
Biosynthesis and immunocytochemical localization of an estrogen-dependent glycoprotein and associated morphological alterations in the sheep ampulla oviduct; Murray MK; Published reports have shown that an M(r) 90,000-92,000 protein is released into the oviductal lumen of the sheep, during estrus at a time corresponding to ovulation and fertilization, where it associates with the embryo . The objectives of this study were (a) to determine whether estradiol-17 beta (E) alone or in combination with progesterone (P) induces the synthesis of the M(r) 90,000-92,000 protein from the ampulla and/or isthmus oviduct; (b) to monitor structural alterations in oviductal epithelial cells associated with the synthesis of this protein; and (c) to generate a polyclonal antiserum to the protein and use the antiserum to verify its cellular location and tissue specificity . Oviductal flushings and explant culture media were obtained from ovariectomized animals treated with E alone or with E plus P . The M(r) 90,000-92,000 protein was present in 3H-leucine- and 3H-glucosamine-labeled culture media of the ampulla (not isthmus) oviduct in animals treated with E alone or with E plus P . The glycoprotein was detected in gels of oviductal flushings obtained from animals treated only with E . A specific polyclonal antiserum to the protein was made and cross-reacted on Western blots of oviductal flushings from E-treated animals and ampulla (not isthmus) oviduct culture media from animals treated with E alone or with E plus P . The secretory apparatus of the epithelial cells of the ampulla oviduct matured and differentiated in response to E . Light and electron microscopic immunocytochemistry localized the M(r) 90,000-92,000 glycoprotein to secretory granules in the nonciliated cells of the ampulla oviduct . Immunoperoxidase reaction product was absent in tissue sections and Western blots of other reproductive and nonreproductive tract tissues obtained from steroid-treated animals . Therefore, the secretory cells of the ampulla oviduct of the sheep synthesize and release an E-induced, oviduct-derived M(r) 90,000-92,000 glycoprotein.

Biochimie, 1992 Nov, 74(11), 975 - 9
Effects of high glucose concentration on survival and respiration of human endothelial cells; Foultier MT et al.; To determine whether or not endothelial cell survival was decreased after incubation with high glucose concentrations in culture media, we studied the influence of D-glucose or L-glucose (a non-metabolizable stereoisomer of D-glucose) on cell survival using the trypan-blue exclusion test . Simultaneously, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to measure both the mitochondrial respiratory chain activity and cell viability . Respiratory chain activity per cell increased when D-glucose concentrations rose but at the same time trypan-blue excluded cells were decreased . Comparison with data in the literature showed that the MTT assay was not reliable for studies involving endothelial cell survival when glucose reduction was affected on these cells . It seems important to check MTT assay reliability carefully when it is used for drugs affecting glucose metabolism, or with other cell types.

Teratology, 1992 Nov, 46(5), 499 - 507
Methionine decreases the embryotoxicity of sodium valproate in the rat: in vivo and in vitro observations; Nosel PG et al.; Methionine provided in the drinking water of pregnant rats injected with sodium valproate reduced the frequency of resorptions but did not improve embryo growth . Rats drinking methionine supplemented water had approximately twice the level of serum-free methionine and consumed only one-half the volume of water of controls . Using whole rat embryo cultures, the simultaneous addition of methionine and sodium valproate to the medium provided no protection from neural tube defects, nor did the addition of methionine to a medium of serum obtained from rats previously dosed with sodium valproate . However, protection from the teratogenic effects of sodium valproate was afforded by methionine when the culture medium was sera from rats consuming methionine and was particularly striking when embryos for culture were taken from pregnant rats that had been consuming methionine . These observations along with those of others indicated the importance of dietary and culture media methionine levels in evaluating experimental and regulatory teratology studies and suggested the possibility that methionine may play an important role in human teratology where multifactorial causes have been implicated in problems such as neural tube closure defects.

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