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),