Biocell, 2000 Dec, 24(3), 247 - 51 Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus; Marcellan ON et al.; Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv . Sprengeri, by pre- and post-zygotic barriers . The latter barrier operates at the endosperm level five days after pollination in A . officinalis x A . densiflorus crosses . To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested . Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions . Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete . Transfer of ovules to other media was necessary to promote embryo development . The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained . Histological studies revealed differentiation of protoderm only . The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed. Anat Histol Embryol, 2000 Dec, 29(6), 363 - 70 Teratogenicity of edoferon kappa A, a molecule derived from salicylate, in cultured rat embryos: differences from salicylate and interaction with free oxygen radical scavenging enzymes; Karabulut AK et al.; The effect of edoferon kappa A (E-KA), a non-specific immunomodulatory and anti-neoplastic chemical substance derived from the methyl form of salicylate (acetyl salicylic acid; ASA), on mammalian embryos was studied and compared to the effects of ASA . Rat embryos were cultured in vitro from 9.5 days of gestation for 48 h . E-KA (0.1-12.8 mg/ml) and ASA (0.1-0.6 mg/ml) were added to the whole rat serum . To investigate the interaction of these molecules with antioxidant agents, the lowest effective concentrations of E-KA (0.6 mg/ml) and ASA (0.3 mg/ml) for all parameters were added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 mumol/ml) . The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations . E-KA and ASA decreased growth and development in a concentration-responsive manner . There was also a concentration-related increase in overall dysmorphology (haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud) . There were no statistically significant differences between the control and embryos grown in the presence of 0.1-0.4 mg/ml E-KA, although the effects of ASA started at a concentration of 0.2 mg/ml . Embryos showed significant growth retardation in all scoring criteria and severe malformations when 0.5-3.2 mg/ml E-KA and 0.3-0.6 mg/ml ASA were added . When SOD was added, there was a significant decrease in the incidence of malformations and growth and developmental parameters were increased but this decrease never reached the control level . We concluded that E-KA has direct toxic effects on the developing embryo but at much higher concentrations than ASA, and the teratogenic effects of these molecules might be related to free oxygen radicals. Xenobiotica, 2000 Nov, 30(11), 1033 - 45 In vitro identification of the cytochrome P450 isoform responsible for the metabolism of alpha-dihydroergocryptine; Althaus M et al.; 1 . The in vitro metabolism of alpha-dihydroergocryptine (DHEC, Almirid), an ergot-derived dopamine agonist for the treatment of Parkinson's disease, has been studied in cultured cell lines following incubation with DHEC . Human hepatocytes as well as two sets of metabolically competent cell lines expressing one single human cytochrome P450 (1A1, 1A2, 1B1, 2A6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4) were used . 2 . Mono- and dihydroxy metabolites of DHEC could only be detected in the culture media of the cell line expressing human cytochrome CYP3A4 . The same metabolites were found in the media of cultured human hepatocytes derived from three different donors . After 24-h incubation with 1 microM DHEC, approximately 60% mono- and approximately 20% dihydroxy metabolites were detected, i.e . approximately 80% of DHEC was metabolized . Further, DHEC demonstrated an inhibitory effect on CYP3A4-mediated testosterone metabolism and additionally could induce CYP3A4 and CYP2E1 mRNA when added at 10 microM to cultured human hepatocytes . 3 . The data suggest that DHEC metabolism in humans is primarily mediated by the CYP3A4 isoform . The results are in accordance with findings derived from other ergot alkaloids. Arch Microbiol, 2000 Dec, 174(6), 393 - 8 Characterisation of coupling products formed by biotransformation of biphenyl and diphenyl ether by the white rot fungus Pycnoporus cinnabarinus; Jonas U et al.; Cells of the white rot fungus Pycnoporus cinnabarinus grown in glucose were able to hydroxylate biphenyl and diphenyl ether, although growth was inhibited by these substrates at concentrations above 250 microM . 2- and 4-Hydroxybiphenyl were detected as products of biphenyl metabolism and 2- and 4-hydroxydiphenyl ether as products of diphenyl ether metabolism in the culture media . After addition of 2-hydroxydiphenyl ether and 2-hydroxybiphenyl to cell-free supernatants containing laccase as the only ligninolytic enzyme, different coloured precipitates were formed . HPLC analysis revealed the formation of additional hydrophobic metabolites with one major product per transformation . Mass spectrometric analysis of the methyl derivatives of the polymer mixture indicated dimers and trimers with different binding types . The main products were identified as dimers with carbon-carbon bonds in para-position to the hydroxyl group of the monomers by mass spectroscopy and nuclear magnetic resonance spectroscopy. Reprod Fertil Dev, 2000, 12(1-2), 7 - 14 Vasoactive intestinal peptide influences hatching of ovine blastocysts; Leoni G et al.; Expanded blastocysts collected from superovulated Sarda ewes were divided at random into four groups for culture in a simple medium that does not support blastocyst hatching (CZB) or a complex medium that is permissive to hatching (TCM 199), with or without vasoactive intestinal peptide (VIP), a known embryo mitogenic peptide . Plasminogen activator (PA) secretion after 24 h of culture, and the number of cells, diameter of blastocysts and hatching rate after 48 h of culture were compared . The results showed an increase in hatching rate (78.6 v . 6.7%; P<0.01), diameter and number of cells (220.89 v . 210.44 microm, P<0.01 and 246 v . 232, P<0.01 respectively) and caseinolytic areas (1.33 v . 0.92 cm, P<0.01) of blastocysts cultured in TCM 199 compared with those cultured in CZB . Supplementation of the culture media with VIP increased these parameters in CZB (P<0.01) and partially in TCM 199 . In particular, cell number, diameter and PA activity were significantly higher (P<0.01) after culture with VIP in both media . Immunoneutralization of exogenous VIP in culture with anti-VIP antibody caused a decrease in the hatching rate (P<0.01) of embryos cultured in medium with VIP, similar to the rate in unsupplemented CZB (P<0.01) . These results suggest a receptor-mediated response . In immunohistochemical studies, VIP was shown to bind receptors in hatched blastocysts demonstrating the VIP-receptor interaction, and VIP receptors of approximately 150 kDa were revealed by electrophoretic studies . In conclusion, ovine preimplantation embryos exhibit VIP receptors, providing a basis for a receptor-mediated influence of VIP in the hatching of ovine blastocysts. Ann N Y Acad Sci, 2000, 923, 9 - 24 The discovery of uteroglobin and its significance for reproductive biology and endocrinology; Beier HM; The discovery of uteroglobin resulted from investigations on the biochemical composition of oviductal and uterine secretions of the rabbit and other mammals . These determinations about physiological composition were urgently requested to prepare culture media for research on early mammalian development in vitro . Discovery of significant proteins during the sixties reflected the laboratory skills of that time . Protein characterization was achieved by isolation via Sephadex gels, electrophoresis on polyacrylamide gels, and finally immunoprecipitation using classical polyclonal antibodies . The molecular biology was not yet established . Uteroglobin could be found as the major protein component of rabbit uterine secretion . From the beginning, it was already identified as an unusually small, spheric uterine secretory molecule without any carbohydrates--hence its name . Uteroglobin was the first mammalian protein that turned out to be progesterone-regulated and, at the same time, released in mg amounts actually in one organ compartment . Moreover, uteroglobin and its gene proved to be a reliable model for the description of progesterone/progesterone receptor complex action at the DNA level . After its original observation in the uterus, however, uteroglobin was detected also in several other organs, for example, the epididymis, the seminal vesicle, and the lung . Initially, it could not be found in the blood, which challenged the hypothesis that uteroglobin specifically should operate by local activation rather than by a humoral or endocrine effect . Later, though, the human uteroglobin molecule, isolated from blood filtrate, was used for detailed structural analyses . The rabbit uteroglobin model certainly was beneficial for reproductive biological research . Experimental interference with steroid hormone regulation during preimplantation presented surprising effects, which led to the discovery of the transposition of the implantation window . The uterine secretion protein patterns, in particular the uteroglobin fraction and the beta-glycoprotein fraction, served as decisive marker profiles to identify the biological stage of the intrauterine microenvironment during preimplantation . This diagnostic procedure, using only protein parameters, enabled us to precisely predict the receptive stage of the endometrium for donated blastocysts to achieve implantation successfully. Ann N Y Acad Sci, 2000, 923, 193 - 201 Tumor necrosis factor alpha stimulation of human Clara cell secretory protein production by human airway epithelial cells; Cowan MJ et al.; Clara cell secretory protein (CCSP) or uteroglobin/CC10 is a product of epithelial cells in a variety of organs including the lung . CCSP has anti-inflammatory properties and may act as an inhibitor of secretory phospholipase A2's . Tumor necrosis factor alpha (TNF-alpha) is capable of inducing the expression of gene products including a variety of cytokines and chemokines in the airway epithelium that may upregulate the airway inflammatory response . Therefore, it was of interest to determine whether this proinflammatory cytokine might also induce the production of a counterregulatory protein such as CCSP, which might modulate the inflammatory response in the airway . Normal human tracheobronchial epithelial cells in primary culture and a human bronchial epithelial cell line (BEAS-2B) were studied . CCSP mRNA levels in BEAS-2B cells were detected by ribonuclease protection assay . CCSP mRNA levels increased in response to TNF-alpha (20 ng/mL) stimulation after 8-36 h, with the peak increase at 18 h . Immunoblotting of CCSP released from BEAS-2B cells into the culture media demonstrated that TNF-alpha induced the synthesis and secretion of CCSP over 8 to 18 h . Similarly, TNF stimulated the release of CCSP from human tracheobronchial epithelial cells in primary culture at 8 and 18 h . The CCSP reporter gene including 801 bases 5' of the transcription start site did not increase transcriptional activity in response to TNF-alpha stimulation . A CCSP mRNA half-life assay indicated that TNF-alpha induced increases in CCSP mRNA at least in part at a posttranscriptional level . Therefore, TNF-alpha induces airway epithelial cell expression of human CCSP and may modulate airway inflammatory responses in this manner. Life Sci, 2000 Dec 1, 68(2), 153 - 63 Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model; Zou J et al.; The ability of resveratrol to inhibit vascular intimal thickening was tested in an experimental model in which endothelial denudation was performed in the normal rabbit iliac artery . Resveratrol (2 approximately 4mg/ kg/d) was administered intragastrically for 5 weeks beginning 1 week before denudation . At the higher concentration of resveratrol, the intimal hyperplasia of injured vascular wall was effectively inhibited; the intimal proliferation index also was significantly less than that in the untreated control group (0.28 +/- 0.07 vs 0.41 +/- 0.13, respectively, p<0.01); the relative luminal area increased from 0.38 +/- 0.06 in the untreated control group to 0.53 +/- 0.10 in the resveratrol treatment group (p < 0.001); and the count of smooth muscle cells in the thickened intima was statistically significantly reduced in the high dose resveratrol treatment group than that in the untreated group (1,115 +/- 510 vs 1,796 +/- 963, respectively, p < 0.05) . Resveratrol added to the culture media of cultured rabbit vascular smooth muscle cells inhibited DNA synthesis in a dose-dependent manner . These results showing that resveratrol is capable of inhibiting intimal hyperplasia of injured artery raise the possibility that this polyphenol might have clinical potential in prevention and treatment of restenosis after angioplasty. Curr Genet, 2000 Dec, 38(5), 291 - 8 Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker; Elrod SL et al.; The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi . Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme . Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase . The hemA gene can therefore be used as a selectable marker for the transformation of A . oryzae. J Androl, 2001 Jan-Feb, 22(1), 96 - 103 Detection of the mouse acrosome reaction by acid phosphatase . Comparison with chlortetracycline and electron microscopy; Pietrobon EO et al.; The sperm acrosome is a uniquely regulated secretory vesicle containing several hydrolase enzymes, including acid phosphatase (AP) . The exocytotic event that releases these enzymes, the acrosome reaction, is required for fertilization in mammals . Different methods have been described in the scientific literature for detection of the acrosome reaction: double and triple stains, fluorescent-lectin stains, monoclonal antibodies against acrosomal antigens (immunodetection techniques), Coomassie blue, differential interference contrast or phase contrast, flow cytometry, and chlortetracycline (CTC) . In contrast, only 1 method to detect AP released by live and reacted sperm has been described in the literature thus far . In this work we compare 2 classical methods, CTC and transmission electron microscopy (TEM), with the assay of AP released from the acrosome . AP released during the acrosome reaction was measured in the culture medium . Enzyme remaining in nonreacted sperm cells was released by Triton X-100 treatment . This enzyme-based methodology shows an increase of AP in the culture media after the acrosome reaction and a corresponding decrease in the detergent-releasable enzyme . The AP assay thus permits the detection of the mouse acrosome reaction and compares well with the CTC and TEM methods . This method is performed on the whole sperm population and so avoids the observer error that is inherent in light microscopic methods. JPEN J Parenter Enteral Nutr, 2001 Jan-Feb, 25(1), 23 - 9 Effects of L-arginine on the proliferation of T lymphocyte subpopulations; Ochoa JB et al.; BACKGROUND: Dietary supplementation of L-arginine as a mechanism to enhance cellular immune response (T lymphocytes), has slowly gained approval, and appears especially important during critical illness . Despite its clinical use, little is known as to the direct effects of L-arginine on the different T lymphocyte subpopulations . METHODS: Lymphocytes were harvested from spleens of C57 B1/6 mice, and proliferation was induced with anti-CD3 in the presence of different concentrations of L-arginine ranging from 0 to 1000 micromol/L . Flow cytometry was used to evaluate the effect of L-arginine on T lymphocyte subpopulations . Interleukin-2 production was measured by ELISA and gene expression by RT-PCR . RESULTS: L-Arginine at or greater than 100 micromol/L significantly enhanced anti-CD3 stimulated T lymphocyte proliferation (p = .01) . L-Arginine was essential for adequate T lymphocyte (CD3+) cellular maturation (p = .01) . Proliferation of Helper T cells (CD4+) was not dependent on L-arginine . In contrast, Cytotoxic T cells (CD8+) showed a dose dependent proliferation in response to L-arginine (p = .01) . Of the CD8+ cells, an increase in the CD45RA negative CD8 positive (memory) T cell subpopulation was observed with the addition of L-arginine . In addition, the number of cell surface CD8 receptors (CD8R) and CD3 receptors (CD3R) increased in the presence of L-arginine (p = .01, p = .04) . Interleukin-2 receptor (IL-2R) expression was not up-regulated by L-arginine . L-Arginine modestly increased IL-2 production and had pronounced effects on its disappearance from the culture media (p < .0001) . Interleukin-2 mRNA expression was not dependent on L-arginine . CONCLUSIONS: The requirements for L-arginine for the proliferation of CD3 stimulated T lymphocytes vary widely, and have to be taken into account when studying the mechanism of how L-arginine enhances cellular proliferation . L-Arginine may increase cellular proliferation by increasing specific receptor expression and the utilization of interleukin-2. Biol Reprod, 2001 Mar, 64(3), 983 - 91 Molecular characterization of bovine prostaglandin G/H synthase-2 and regulation in uterine stromal cells; Liu J et al.; Prostaglandin G/H synthase (PGHS) is a key rate-limiting enzyme in the prostaglandin biosynthetic pathway, and prostaglandins play a central role in the control of the reproductive cycle . The objectives of this study were to clone and characterize the primary structure of bovine PGHS-2 and to study its regulation in uterine stromal cells in vitro . The bovine PGHS-2 cDNA was cloned by a combination of reverse transcription-polymerase chain reaction and cDNA library screening . Results showed that the complete bovine PGHS-2 cDNA is composed of a 5'-untranslated region of 128 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1565 bp containing multiple repeats (n = 11) of the Shaw-Kamen sequence 5'-ATTTA-3' . The open reading frame encodes a 604-amino acid protein that is 86-97% identical to other mammalian PGHS-2 homologs . The regulation of PGHS-2 mRNA and protein was studied in primary cultures of bovine uterine stromal cells stimulated with phorbol 12-myristate 13-acetate (PMA; 100 nM) . Northern and Western blot analyses reveal a marked induction in PGHS-2 transcript (4.0 kilobases) and protein (M(r) = 72 000) after 3-12 h of PMA stimulation (P < 0.05) . However, this induction was transient in nature as levels of PGHS-2 mRNA and protein returned to basal levels after 24 h of PMA stimulation . In contrast, PMA had no effect on levels of PGHS-1 (P > 0.05) . The PMA-dependent induction of PGHS-2 was associated with a significant increase in prostaglandin E2 secretion in the culture media (P < 0.05) . To study promoter activity of the 5'-flanking DNA region of the bovine PGHS-2 gene, the genomic fragment -1574/-2 (+1 = transcription start site), as well as a series of 5'-deletion mutants, were fused upstream of the firefly luciferase gene and transiently transfected into primary cultures of bovine uterine stromal cells . Results showed that a first promoter region located between -1574 and -492 and a second region between -88 and -39 appear to play important roles in PMA-dependent regulation of PGHS-2 promoter activity in bovine uterine cells . Thus, this study characterizes for the first time the structure of the bovine PGHS-2 transcript and the deduced amino acid sequence of its encoded protein and establishes an in vitro model to study the regulation of PGHS-2 gene expression in bovine uterine tissue. Regul Pept, 2001 Apr 2, 98(1-2), 49 - 54 Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures; Galfi M et al.; The effects of dopamine (DA) or DA-active drugs on the synthesis of neurohypophyseal (NH) hormones were studied in 13-14 day cultures of isolated NH tissue from rats . The following DA-active compounds were used (10(-6) M in each medium): DA, apomorphine (APM), Pro-Lys-Gly (PLG), butaclamol (B), haloperidol (HP), chlorpromazine (CPZ) and sulpiride (SP) . The oxytocin (OT) and vasopressin (VP) contents of the condensed media were determined by RIA after a 1 or 2 h incubation . Significantly increased contents of OT and VP were detected in the tissue culture media following DA, APM or PLG administration . This elevation of NH hormone production could be blocked by previous administration of B or the DA receptor antagonists HP, CPZ or SP . The application of B after DA agonists proved ineffective . The results indicate that NH hormone production can be directly influenced by the DA-ergic system . The DA-ergic control of NH hormone secretion in rats can occur independently of the hypothalamus, at the level of the posterior pituitary. Biochem Biophys Res Commun, 2001 Feb 16, 281(1), 180 - 5 Osteoclasts secrete the chemotactic cytokine mim-1; Falany ML et al.; Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin . In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically . Peptide digests of the protein were analyzed by mass spectroscopy . The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein) . Mim-1 is expressed specifically by hematopoietic cells and has no known function . It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes . Western analysis showed that osteoclasts secrete mim-1 into culture media . Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells . Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling. Nitric Oxide, 2001 Feb, 5(1), 62 - 71 A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite; Miranda KM et al.; Numerous methods are available for measurement of nitrate (NO(-)(3)) . However, these assays can either be time consuming or require specialized equipment (e.g., nitrate reductase, chemiluminescent detector) . We have developed a method for simultaneous evaluation of nitrate and nitrite concentrations in a microtiter plate format . The principle of this assay is reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction . This assay is sensitive to 0.5 microM NO(-)(3) and is useful in a variety of fluids including cell culture media, serum, and plasma . S-Nitrosothiols and L-arginine derivatives were found to be potential interfering agents . However, these compounds are generally minor constituents of biological fluids relative to the concentration of nitrate/nitrite . This report introduces a new, convenient assay for the stable oxidation products of nitrogen oxide chemistry in biological samples . Ann Surg, 2001 Feb, 233(2), 287 - 91 Fibroblasts from the transversalis fascia of young patients with direct inguinal hernias show constitutive MMP-2 overexpression; Bellon JM et al.; OBJECTIVE: To determine the expression pattern of certain metalloproteinases (MMPs) known to be involved in the degradation of the extracellular matrix in cultured fibroblasts from the transversalis fascia (TF) of patients with inguinal hernia . SUMMARY BACKGROUND DATA: Inguinal hernia is a common pathology, the cause of which remains unknown . It is, however, clear that the TF is one of the anatomical structures that may impede the formation of hernias, and particularly the direct type of hernia . In previous studies the authors found enhanced MMP-2 expression in TF specimens in vivo . The persistence of increased expression in cultured fibroblasts might support the idea of a genetic defect as the cause for this pathology . METHODS: Fibroblasts from the TF of patients with direct and indirect inguinal hernia were cultured and compared with those obtained from control TF in terms of MMP (MMP-2 and MMP-9) expression . RESULTS: Significant active MMP-2 expression was shown by TF fibroblasts from young patients with direct hernias . These findings were confirmed by immunosorbent assay, immunoblotting, and zymography of the fibroblast culture media . No MMP-9 expression was detected . CONCLUSION: These results indicate that MMP-2 may be involved in the TF matrix degradative process in patients with direct hernia . The persistence of changes in MMP-2 levels in the cell cultures appears to suggest a genetic defect or irreversible change as the origin of this pathology rather than environmental factors, which may later participate in the development of the hernial process. Neurosci Lett, 2001 Mar 2, 300(1), 54 - 8 Chronic morphine treatment inhibits oxytocin release from the supraoptic nucleus slices of rats; Li J et al.; Effect of chronic morphine treatment on oxytocin (OT) release from the long term-cultured organotypic slice of the supraoptic nucleus (SON) was investigated using radioimmunoassay . The co-localization of oxytocin and mu-opioid receptor in neurons within the SON was observed with the double-labeled methods of in situ hybridization combined with immunohistochemistry . After exposure to morphine for 6days, the OT levels in culture media were significantly decreased . Naloxone caused much greater release of OT in chronic morphine treatment group than in controls . Naloxone has no effect after acute morphine treatment . 90% of OT-ir (immunoreactive) neurons expressed mu-opioid receptor mRNA in the SON and 45% of the neurons that expressed mu-opioid receptor mRNAs were OT-ir neurons . These results indicated that the neurons within SON could develop dependence on morphine in vitro, and these effects might be exerted via mu-opioid receptor in oxytocin neurons of the SON. Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1655 - 60 From cell death to embryo arrest: mathematical models of human preimplantation embryo development; Hardy K et al.; Human preimplantation embryos exhibit high levels of apoptotic cells and high rates of developmental arrest during the first week in vitro . The relation between the two is unclear and difficult to determine by conventional experimental approaches, partly because of limited numbers of embryos . We apply a mixture of experiment and mathematical modeling to show that observed levels of cell death can be reconciled with the high levels of embryo arrest seen in the human only if the developmental competence of embryos is already established at the zygote stage, and environmental factors merely modulate this . This suggests that research on improving in vitro fertilization success rates should move from its current concentration on optimizing culture media to focus more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages. Mol Reprod Dev, 2001 Mar, 58(3), 269 - 75 Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media; Gandhi AP et al.; Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium . Currently, little is known regarding porcine embryo metabolism . The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems . Oocytes were matured and fertilized according to standard protocols . Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi) . Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry . Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively) . Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively) . Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively) . Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h) . Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70) . When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05) . In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05) . As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos . Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages . Clin Microbiol Infect, 2000 Jun, 6(6), 303 - 7 Use of short-term culture for identification of Mycobacterium avium subsp . paratuberculosis in tissue from Crohn's disease patients; Schwartz D et al.; OBJECTIVE: To investigate the role of Mycobacterium avium subsp . paratuberculosis (MAP) in Crohn's disease (CD), using short-term mycobacterial culture media . METHODS: Sixty-three tissue specimens from 27 CD patients and 36 controls were processed and inoculated into a modified 7H9 broth base medium and incubated at 37 degrees C and 5% CO2 for up to 1 year . Acid-fast staining, determination of mycobactin dependency, PCR analysis using two IS900-derived oligonucleotides and hybridization with an internal probe were performed . RESULTS: MAP was present in six of seven (86%) surgically resected tissue samples and in four of 20 (20%) biopsies, with an overall 37% from CD patients, as compared to two of 36 (5.6%) of control specimens . The presence of MAP in Mycobacterial Growth Indicator Tube (MGIT) cultures was detected within 10-12 weeks for surgically resected tissue and after 40 weeks for biopsy specimens, with no MAP growth detected in 12B* Bactec cultures . CONCLUSIONS: Because MAP was present in 86% of resected tissue compared to 20% of biopsy specimens from CD patients, we speculate that MAP resides in the submucosal layer closer to the active part of the ulcer rather than on the surface of the mucosal cells . Thus, surgically resected tissue cultured in MGIT medium is a favorable protocol for rapid cultivation of MAP and for investigating its role in CD pathogenesis . The data support the mycobacterial role in CD pathogenesis. Bone, 2001 Jan, 28(1), 21 - 8 Glucose-induced inhibition of in vitro bone mineralization; Balint E et al.; Patients with diabetes tend to have an increased incidence of osteopenia that may be related to hyperglycemia . However, little is known about how glucose may alter bone formation and osteoblast maturation . To determine whether glucose affects osteoblastic calcium deposition, MC3T3-E1 cells were incubated in media containing either a normal (5.5 mmol/L) or high glucose concentration (15 mmol/L) or mannitol (15 mmol/L), and bone nodule formation was examined . Net calcium flux was measured thrice weekly and cumulative calcium uptake was determined . Compared with control incubations, glucose significantly inhibited daily and cumulative calcium uptake into the nodules . At the time of matrix maturation, cultures undergo a rapid phase of increased calcium deposition; this was significantly inhibited by the presence of glucose . Total calcium uptake, determined by acid digestion, was also significantly inhibited by glucose . Area and number of nodules were quantitated at the end of the incubation period (day 30) by staining with Alizarin Red S calcium stain . Compared with both control and mannitol-treated cultures, the number of nodules was increased by incubation with glucose . Furthermore, both the average total nodular area and calcified nodular area of large nodules were increased by glucose . Cellular proliferation as well as the release of markers of osteoblast activity (osteocalcin and alkaline phosphatase) were determined at the end of the experimental period (day 30) . Cellular proliferation and alkaline phosphatase activity was significantly increased in the presence of glucose, however, the release of osteocalcin into culture media was similar in all three groups . In conclusion, the present study shows that elevated glucose concentration present throughout the development of murine osteoblasts stimulates cellular proliferation while inhibiting calcium uptake . The result of glucose inhibition of calcium uptake suggests that bone could be structurally altered in diabetes. Exp Gerontol, 2001 Jan, 36(1), 65 - 78 Is increased arachidonic acid release a cause or a consequence of replicative senescence? Lorenzini A, Hrelia S, Bordoni A, Biagi P, Frisoni L, Marinucci T, Cristofalo VJ. Arachidonic acid (AA) has been related to both stimulation and inhibition of cellular proliferation . During replicative senescence of human fibroblasts, increased levels of AA have been thought to play a causal role in the limited proliferative capacity of the cells . To clarify the role of AA in the proliferation of normal fibroblasts and in cellular senescence, we examined uptake from and release of AA into the culture media and its effects on DNA synthesis . Our results indicate that some aspects of AA metabolism in normal human fibroblasts aged in culture are significantly different in comparison to early passage cells . Particularly, AA release following different mitogenic stimulation is higher in senescent than in young cells . Notwithstanding this significant difference, AA, at the concentration used, has no inhibitory effect on fibroblast DNA synthesis . Moreover AA and prostaglandins are responsible for the proliferative block in neither senescent cells nor mediate ceramide inhibition of DNA synthesis . So our results suggest that the increasing AA release is not causal, but rather the result of in vitro aging. Biochem Biophys Res Commun, 2001 Jan 26, 280(3), 831 - 5 Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells; Brandstrom H et al.; Osteoprotegerin (OPG) is a soluble receptor for receptor activator of NF kappa B-ligand, a factor required for osteoclastogenesis . OPG secreted from bone marrow stromal cells is believed to inhibit osteoclast differentiation and several agents known to influence bone resorption have been demonstrated to regulate mRNA levels of OPG . In this report we have investigated the secretion of OPG protein from primary cultures of human bone marrow stromal cells . An ELISA was developed for measuring the concentration of OPG in culture medium . OPG secretion was decreased by 50% when the human bone marrow stromal cells were treated with 1 microM of prostaglandin E(2), possibly through activation of the protein kinase A-pathway since stimulation of protein kinase A by forskolin also inhibited OPG secretion . Treatment with phorbol 12,13 di butyrate, an activator of the protein kinase C-pathway, potently stimulated the secretion of OPG from human bone marrow stromal cells . The cells were also stimulated with inflammatory mediators and glucocorticoids . Treatment with interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) stimulated OPG secretion to 500% and 400% of control whereas dexamethasone decreased OPG production by 40% . In conclusion, an ELISA measuring OPG in cell culture media was developed . Using this ELISA, the amount of OPG secreted from human bone marrow stromal cells was clearly detectable, and the secretion of OPG-protein was potently regulated by prostaglandin E(2), forskolin, phorbol 12,13 di butyrate, IL-1 alpha, TNF-alpha, and dexamethasone . Cytokine, 2001 Feb 7, 13(3), 148 - 54 Influence of glutamine on cytokine production by human gut in vitro; Coeffier M et al.; BACKGROUND: glutamine modulates cytokine production by immune cells in vitro and protects the gut from experimental enterocolitis, but data on the effect of glutamine on cytokine production in human gut are lacking . AIM: to assess the effect of glutamine pre-treatment in vivo and in vitro on cytokine production by intestinal mucosa . METHODS: nine fasted volunteers received either enteral glutamine or saline over 6 h in a cross-over design . Duodenal biopsies were cultured for 24 h with or without glutamine . Cytokine content of culture media was analysed by ELISA, and the expression of cytokine mRNA in biopsies was assessed by semi-quantitative RT-PCR . Results: glutamine given in vivo and in vitro significantly decreased IL-6 {1.4 (0.8-8.5) vs 8.9 (1.0-43.9)} and IL-8 production {5.8 (0-51.4) vs . 53.0 (2.5-114.6), pg/mg wet tissue}, median (range), both P< or =0.01, in comparison to no glutamine experiments . Glutamine did not influence IL-4 production . IL-1beta, IL-10 and TNF-alpha were not detectable in culture media . The expression of any cytokine mRNA was not influenced by glutamine . CONCLUSIONS: glutamine reduces pro-inflammatory cytokine production by human intestinal mucosa, probably by a post-transcriptional pathway . Glutamine could be useful to modulate inflammatory conditions with imbalanced cytokine production . Anal Biochem, 2001 Feb 15, 289(2), 231 - 8 A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts; Tombaccini D et al.; Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media . CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection . The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample . Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors . This will provide a pattern that might reflect more closely the real CP levels in intact cells . The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions . Mol Hum Reprod, 2001 Feb, 7(2), 195 - 200 Soluble HLA-G influences the release of cytokines from allogeneic peripheral blood mononuclear cells in culture; Kanai T et al.; Exquisitely regulated cytokine balance during early pregnancy is thought to be necessary for promoting survival of the fetal allograft . Our previous studies have demonstrated that membrane-bound human leukocyte antigen (mHLA-G) expressed on trophoblasts is one of the key factors in regulating cytokine balance by shifting the Th1/Th2 balance toward Th2 polarization, a favourable milieu for the maintenance of pregnancy . Given that trophoblasts secrete soluble HLA-G (sHLA-G), we examined its biological roles in comparison with mHLA-G . We cultured peripheral blood mononuclear cells (PBMC) with either the HLA-A and -B-deficient B lymphoblast cell line (721.221 cells) or the same cell line transfected with mHLA-G (721.221-G1 cells), in the presence or absence of recombinant sHLA-G . Cytokine concentrations in the culture media were determined by enzyme-linked immunosorbent assay . In contrast to mHLA-G protein, sHLA-G stimulated the release of tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, whereas it reduced the release of interleukin (IL)-3, regardless of the presence of the presence of a stimulatory effect of the mHLA-G-expressing cells . Although mHLA-G reduced the release of IL-4, sHLA-G did not have any effect . Conversely, sHLA-G stimulated the release of IL-10 whereas mHLA-G was without effect . These results suggest that sHLA-G regulates the release of cytokines from PBMC chiefly by counterbalancing mHLA-G, and thereby may play a role in maintaining pregnancy. Drug Metab Dispos, 2001 Feb, 29(2), 111 - 20 Epidermal growth factor regulation of female-dependent CYP2A1 and CYP2C12 in primary rat hepatocyte culture; Garcia MC et al.; In the present study, we describe the effects of medium composition in primary cultures of rat hepatocytes on the expression of two major constituent female-dependent CYP isoforms, CYP2C12 and CYP2A1 . When female rat hepatocytes were cultured with the serum-free medium HepatoZYME, currently used to attain long-term maintenance of hepatocyte phenotypic expression, CYP2C12 mRNA and protein levels were markedly suppressed, despite the constant presence of growth hormone, the essential regulator of liver CYP2C12 . Conversely, rat hepatocytes cultured in the serum-free medium Dulbecco's modified Eagle's medium-F12K, also supplemented with growth hormone, sustained near normal expression levels of CYP2C12 mRNA and protein for the 7 days of observations . Although media composition had no significant effect on mRNA expression of CYP2A1, protein content decreased dramatically in hepatocytes cultured with HepatoZYME medium . We were able to demonstrate the plasticity of the cells by restoring/suppressing the expression of CYP2C12 and CYP2A1 mRNA by reverting the culture conditions . Addition of the mitogen epidermal growth factor present in the HepatoZYME formulation to the Dulbecco's modified Eagle's medium-F12K culture media appreciably decreased expression of both CYP2C12 and CYP2A1 in female hepatocytes, while briefly sustaining levels of the cyclin inhibitor p21 . Lastly, reduced CYP protein content observed in hepatocytes cultured with epidermal growth factor was not the result of an absence or reduction in the CCAAT/enhancer-binding protein alpha, a requisite transcription factor for CYP2C12 expression. Br J Ophthalmol, 2001 Feb, 85(2), 147 - 53 Tear film MMP accumulation and corneal disease; Smith VA et al.; BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) accumulate in the tears of patients with active peripheral ulcerative keratitis (PUK) but it is unknown whether these enzymes have a central role in disease progression . The aims of the present investigation were to determine the source of these enzymes and to ascertain whether their accumulation in tears is a phenomenon specific to PUK or a general feature of other anterior segment diseases . METHODS: The experimental samples were obtained from the culture media of conjunctival and corneal epithelial cells, from fractionated blood plasma and leucocytes of healthy subjects and patients with rheumatoid arthritis, and from the tears of healthy subjects and patients with a variety of anterior segment diseases . The MMPs of all samples were visualised by zymography and tear samples were assayed using nitrophenol acetate and an MMP-9 susceptible quenched fluorescent peptide as substrate . RESULTS: The major MMPs that accumulate in the tears of patients with rheumatoid arthritis with active ocular disease are MMP-9 and a species of M(r) 116,000 . By comparing the zymographic activity profiles of the gelatinases present in the samples obtained, it was deduced that the main source of these MMPs was granulocytes . Their accumulation in tears was not unique to patients with PUK; detectable amounts of the enzymes also occurred in the tears of patients with keratoconus with associated atopic disease, patients undergoing treatment for herpetic eye disease, and patients with systemic and non-systemic dry eye disease . CONCLUSION: The MMPs that accumulate in tears are mainly derived from granulocytes . This may be effected by autoimmune diseases that involve ocular tissue or by ocular diseases that induce an inflammatory response. Microbiology, 2001 Feb, 147(Pt 2), 459 - 71 Differential expression of mycobacterial proteins following phagocytosis by macrophages; Monahan IM et al.; Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood . Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential . In an attempt to define M . bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1 . This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock . Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M . tuberculosis-infected sera . Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf) . Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M . tuberculosis from vaccination with BCG. J Clin Microbiol, 2001 Feb, 39(2), 533 - 8 Detection and heterogeneity of herpesviruses causing Pacheco's disease in parrots; Tomaszewski E et al.; Pacheco's disease (PD) is a common, often fatal, disease of parrots . We cloned a virus isolate from a parrot that had characteristic lesions of PD . Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1 . Five primer sets were developed from these sequences . The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD . The primer sets amplified DNA from all but one sample . Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses . A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%) . A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses . PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses. Hum Reprod, 2001 Feb, 16(2), 300 - 5 Maturation of rhesus monkey oocytes in chemically defined culture media and their functional assessment by IVF and embryo development; Zheng P et al.; This study compared success of in-vitro maturation of rhesus monkey oocytes in protein-free versus serum-containing culture systems, assessed by embryo development subsequent to IVF . Four media were tested: (i) modified Connaught Medical Research Laboratories medium (mCMRL-1066); (ii) hamster embryo culture medium-10 (HECM-10); (iii) control: mCMRL-1066 + 20% bovine calf serum (BCS); (iv) HECM-10 + 20% BCS . Immature oocytes from FSH-stimulated rhesus monkeys were allocated among the media containing ovine FSH (5 microg/ml) and LH (10 microg/ml) and cultured for 36-40 h . Metaphase II ova were inseminated and putative zygotes were cultured in mCMRL-1066 + 20% BCS until development arrested . Ova matured in all four media had similar (P> 0.05) potential to initiate (66, 67, 82 and 69% respectively) and complete meiotic maturation (60, 50, 76 and 57% respectively) . Inseminated ova in all groups had similar potential to be fertilized (86, 83, 84 and 90% respectively), cleave (71, 83, 76 and 90% respectively) and develop to the blastocyst stage (19, 17, 16 and 30% respectively) . These results indicate for the first time that primate oocytes can be successfully matured in protein-free medium, with subsequent blastocyst development, comparable to responses in complex medium with serum . This finding will facilitate studies on mechanisms regulating primate oocyte maturation. Circulation, 2001 Feb 6, 103(5), 634 - 7 Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia; Kawamoto A et al.; BACKGROUND: We investigated the therapeutic potential of ex vivo expanded endothelial progenitor cells (EPCs) for myocardial neovascularization . METHODS AND RESULTS: Peripheral blood mononuclear cells obtained from healthy human adults were cultured in EPC medium and harvested 7 days later . Myocardial ischemia was induced by ligating the left anterior descending coronary artery in male Hsd:RH-rnu (athymic nude) rats . A total of 10(6) EPCs labeled with 1,1'-dioctadecyl-1 to 3,3,3',3'-tetramethylindocarbocyanine perchlorate were injected intravenously 3 hours after the induction of myocardial ischemia . Seven days later, fluorescence-conjugated Bandeiraea simplicifolia lectin I was administered intravenously, and the rats were immediately killed . Fluorescence microscopy revealed that transplanted EPCs accumulated in the ischemic area and incorporated into foci of myocardial neovascularization . To determine the impact on left ventricular function, 5 rats (EPC group) were injected intravenously with 10(6) EPCs 3 hours after ischemia; 5 other rats (control group) received culture media . Echocardiography, performed just before and 28 days after ischemia, disclosed ventricular dimensions that were significantly smaller and fractional shortening that was significantly greater in the EPC group than in the control group by day 28 . Regional wall motion was better preserved in the EPC group . After euthanization on day 28, necropsy examination disclosed that capillary density was significantly greater in the EPC group than in the control group . Moreover, the extent of left ventricular scarring was significantly less in rats receiving EPCs than in controls . Immunohistochemistry revealed capillaries that were positive for human-specific endothelial cells . CONCLUSIONS: Ex vivo expanded EPCs incorporate into foci of myocardial neovascularization and have a favorable impact on the preservation of left ventricular function. J Virol, 2000 May, 74(9), 4244 - 52 A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice; Chang GJ et al.; Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter . COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media . Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX . All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization . JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400 . Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14 . Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50, 000-PFU JEV intraperitoneal challenge . These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine. Invest Ophthalmol Vis Sci, 2000 Apr, 41(5), 1176 - 80 Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells; Lyons TJ et al.; PURPOSE: Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes . Similar modifications occurring in vivo in diabetes may contribute to retinopathy . The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL . METHODS: Minimal in vitro modification of LDL (3 days, 37 degrees C) was achieved with glucose (0, 50 mM), under antioxidant conditions (for N-LDL, G-LDL), or under mild oxidant conditions (for MO-, GO-LDL) in the presence/absence of aminoguanidine (0, 1, 10, 100 microM) . Glucose and aminoguanidine were then removed by dialysis . Confluent bovine retinal capillary endothelial cells (n = 13) and pericytes (n = 14) were exposed to LDL (100 mg/l) for 3 days, with and without aminoguanidine (100 microM) in media . Cell counts were determined by hemocytometer . RESULTS: A decrease in cell counts after exposure to modified compared with N-LDL was confirmed (P < 0.001) but was significantly mitigated if LDL had been modified in the presence of aminoguanidine (P < 0.001) . Aminoguanidine was as effective at 1 microM as at the higher concentrations . Aminoguanidine (100 microM) present in culture media conferred no additional protection, and showed slight evidence of toxicity . Aminoguanidine present during LDL modification had no effect on measured glycation or oxidation products, or on LDL oxidizability . CONCLUSIONS: Very low concentrations of aminoguanidine mitigate toxicity of LDL exposed to stresses that simulate the diabetic environment . This action may contribute to the beneficial effects of aminoguanidine observed in experimental diabetic retinopathy. Arch Virol, 2000, 145(2), 333 - 51 The extracellular part of glycoprotein E of bovine herpesvirus 1 is sufficient for complex formation with glycoprotein I but not for cell-to-cell spread; Tyborowska J et al.; Glycoproteins gE and gI of bovine herpesvirus 1 (BHV-1) are type I transmembrane proteins that can form a complex that is involved in cell-to-cell spread mechanisms . The extracellular domains of both proteins have cysteine-rich regions that are also found in the homologous proteins of other alpha-herpesviruses . The extracellular domain of gE has two conserved cysteine-rich regions: C1 and C2 . The other conserved regions in gE are located between C2 and transmembrane region and in the cytoplasmic domain of gE . We studied the complex formation between gE and gI using a series of truncated gE proteins and a full length form and a secreted form of gI . All proteins were expressed in recombinant baculoviruses . To analyse the complex formation between these polypeptides we used monoclonal antibodies (MAbs 67 and 75) that specifically react with the gE/gI complex and not with separately expressed glycoproteins gE and gI alone . This analysis showed that the BHV-1 gE/gI complex can be formed in insect cells after a co-infection with baculoviruses expressing gE and gI in their full length form . When secreted forms of gE and gI were expressed after co-infection, the gE/gI complex was still formed and could also be detected in the tissue culture medium . This gE/gI complex was also formed after mixing the tissue culture media of insect cells expressing the secreted form or gE or gI separately . The smallest part of gE that still formed a complex is encoded by the first 246 residues of gE . This extracellular domain contains only the C1 region, showing that the C2 region is not essential for gE/gI complex formation . Shorter forms of gE encoding the C1 region did not form a detectable complex . We also found that the formation of gE/gI complex is not sufficient for normal cell-to-cell spread of BHV-1 . A recombinant BHV-1 gE TM-virus, expressing a truncated glycoprotein E from which the transmembrane and cytoplasmic domain were removed, forms plaques as small as a gE null mutant. J Immunol Methods, 2001 Jan 1, 247(1-2), 217 - 24 Specific affinity depletion of cell adhesion molecules and growth factors from serum; Underwood PA et al.; Serum is a common component of most in vitro cell culture media, particularly of primary cells . Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement . We describe a combined affinity protocol for removing vitronectin and fibronectin from serum . This protocol can also be used to purify these molecules . We also describe the removal of growth-promoting elements using heparin-Sepharose . As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence . This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems . It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors. J Cardiovasc Pharmacol Ther, 2000 Oct, 5(4), 313 - 22 Cocaine increases beta-myosin heavy-chain protein expression in cardiac myocytes; Henning RJ et al.; BACKGROUND: As many as 47% of chronic cocaine users develop cardiac ventricular hypertrophy . The presence and degree of cocaine-induced ventricular hypertrophy is not correlated with the use of other substances of abuse such as alcohol or cigarettes . Moreover, this hypertrophy occurs in individuals without sustained increases in arterial blood pressure or heart rate, or increases in the plasma concentration of renin, aldosterone, norepinephrine, or cortisol . Therefore, we investigated whether cocaine, in concentrations commonly found in cocaine users, has any direct effects on the protein content in cardiac ventricular myocytes . We compared the effects of cocaine with norepinephrine, which increases the total protein content, especially beta-myosin heavy-chain contractile protein (beta-MHC), in cardiac ventricular myocytes . METHODS: Experiments were performed on 30-day-old rat ventricular myocytes suspended in culture media and cultured in flasks . In 12 suspension-culture experiments, cocaine or norepinephrine, in doses of 0 (control) or 10(-6) mol/L was added to each culture and the cells were harvested on day 5 . In 16 flask-culture experiments, cocaine or norepinephrine was added to each culture on day 7 in doses of 0 (control-vehicle), 10(-7), or 10(-6) mol/L and the cells were harvested on day 10 . The total protein content and the myosin protein expression of the myocytes in each culture were determined . Juvenile and adult rat cardiac myosin protein is predominately alpha-myosin heavy-chain protein (alpha-MHC), whereas beta-MHC occurs primarily in fetal rat hearts . RESULTS: In the suspension-culture experiments, cocaine, 10(-6) mol/L, increased the cardiomyocyte total protein concentration by 29% +/- 2% (P <.001) and the beta-MHC expression by 81% +/- 10% (P <.01) in comparison with the control myocytes . Cocaine slightly decreased cardiomyocyte alpha-MHC . Norepinephrine increased the total protein concentration by 21% +/- 3% (P <.001) and the beta-MHC expression by 59% +/- 10% (P <.01), but did not increase alpha-MHC expression . In the flask-culture experiments, cocaine, 10(-6) mol/L, maximally increased the total protein concentration by 28% (P <.001), the protein/cell ratio by 57% +/- 10% (P <.01), and the beta-MHC expression by 85% +/- 8% (P <.01) . Cocaine slightly decreased alpha-MHC . Norepinephrine, 10(-6) mol/L, maximally increased the total protein concentration by 35%, the protein/cell ratio by 63% +/- 9% (P <.01), and the expression of beta-MHC by 78% +/- 11% (P < . 01) . Norepinephrine did not increase alpha-MHC expression . In 18 separate flask-culture experiments, cocaine, 10(-6) mol/L, was added to the cardiomyocyte cultures after the addition of phentolamine (n = 9), in concentrations of 10(-7) to 10(-5) mol/L, or metoprolol (n = 9), in concentrations of 10(-7) to 10(-5) mol/L . Neither phentolamine nor metoprolol inhibited the cocaine-induced increase in cardiomyocyte total protein content or the expression of beta-MHC . CONCLUSION: Cocaine, similar to norepinephrine, significantly increases the total protein content and the expression of beta-MHC in cardiac ventricular myocytes . In this manner, cocaine may cause cardiac ventricular hypertrophy . This process is not inhibited by alpha- or beta-adrenergic receptor blockade. Neurol Res, 2000 Dec, 22(8), 797 - 801 Elevated transferrin concentration in cerebral spinal fluid after subarachnoid hemorrhage; Takenaka KV et al.; Calcium-elevating protein cross-reacted with anti-human transferrin (TF) was purified in cerebrospinal fluid (CSF) after subarachnoid hemorrhage (SAH) . The concentration of TF in CSF after SAH was measured, and the effects of TF on cultured smooth muscle cells (SMCs) were evaluated in order to understand the relationship between TF and cerebral vasospasm . Cisternal CSF samples were collected from 12 patients (seven men and 13 women) with SAH due to the rupture of a cerebral aneurysm, and eight control subjects . The patients were divided into two groups: (1) those presenting no clinical and radiological evidence of vasospasm (the non-vasospasm group; three men and 10 women, mean age 54.7 years), and (2) those presenting evidence of vasospasm (the vasospasm group; four men and three women, mean age 58.3 years) . The concentration of TF in CSF was measured using Speriol micro-transferrin measure assay method . Nitrite accumulation in the culture media of SMCs incubated with TF was determined by diazotization method . The mRNA levels of inducible isoform of NOS (iNOS) in SMC incubated with TF were measured by the reverse transcription-coupled polymerase chain reaction method . Levels of TF were markedly different in the SAH and the control subjects . In the control group, all subjects had no detectable quantity of TF . In contrast, all patients after SAH had quantifiable TF in their CSF . Moreover, there was a significant difference between the vasospasm group and the non-vasospasm group in TF levels (p < 0.05) . In the vasospasm group, the average value was 10.43 +/- 2.8 mg dl-1 . In the non-vasospasm group, the average was 3.69 +/- 0.31 mg dl-1 . The nitrite content in the culture medium incubated with TF was three times the content of control . TF also induced iNOS mRNA in SMC . This study demonstrated that an elevation of TF concentration in CSF after SAH was detected and iNOS mRNA in SMCs was also induced by TF . This may be involved in some roles of the development of the pathological series of events after SAH, including vasospasm. Enferm Infecc Microbiol Clin, 2000 Nov, 18(9), 439 - 44 {Negative effect of the components of the lysis-centrifugation system in the growth of mycobacteria in MGIT and Septi-Chek AFB liquid media}; Gamboa F et al.; BACKGROUND: The lysis-centrifugation system (Isolator system) is a technique with excellent results in the recovery of mycobacteria from blood specimens . This system consists mainly of saponin (SAP), polypropylenglycol (PPG), and sodium polianthol sulfonate (SPS) . The objective of this work was to determine the effect of SAP, PPG, and SPS on the growth of Mycobacterium avium, M . kansasii, M . tuberculosis, and M . xenopi in fluid culture media MGIT and Septi-Chek AFB . METHODS: Two concentrations each of SAP, PPG, and SPS were prepared, and were added in 0.1 ml amounts (alone, in pairs and in combination) to fluid media MGIT and Septi-Chek AFB . Fluid culture media were then in individually inoculated with two different concentrations (10(3) and 10(5) CFU/ml) of each of the four mycobacterial strains used in this study . Culture media were incubated at 37 degrees C and were checked for growth daily . RESULTS: SAP, PPG, and SPS did not inhibit growth of mycobacteria but growth of these strains was indeed retarded (a lengthier time was required for detection of bacterial growth compared with the positive control) . Final concentrations of SAP, PPG, and SPS which retarded mycobacterial growth varied, depending upon species, mycobacterial inoculum size, and fluid culture media used . CONCLUSIONS: Components included in the lysy-centrifugation system (SAP, PPG, and SPS), either alone or in combination retarded growth of M . avium, M . kansasii, M . tuberculosis, and M . xenopi in 10(3) and 10(5) CFU/ml concentrations in fluid culture media MGIT and Septi-Chek AFB . These results suggest that strategies should be adopted to decrease the concentrations of these three components, present in the sediment of the processed blood by the Isolator System, which eventually are going to be added to fluid media MGIT and Septi-Check AFB. Drug Alcohol Depend, 2001 Jan 1, 61(2), 155 - 62 Cytotoxic effect of alcohol-withdrawal on primary cultures of cortical neurones; Nagy J et al.; Physical dependence on alcohol was observed previously at the cellular level in cultured IM-9 human lymphoblast cells . To answer the question whether physical dependence can also develop in neurones and to investigate the neuronal processes involved in the development of alcohol dependence and withdrawal symptoms, cultures of cortical neurones were adapted to alcohol . Morphological characteristics of neurones were not altered during the chronic (3-day) repeated (once per day) ethanol (50-100 mM) treatment, whereas obvious signs of neuronal damage were seen after the following 24 h of alcohol-withdrawal . The extent of the damage, quantitated by measuring the release of lactate dehydrogenase (LDH) into the culture media, was dependent on the concentration of ethanol in the medium during adaptation . LDH-release induced by alcohol-withdrawal was significantly reduced by re-addition of ethanol, as well as by administration of non-competitive (MK-801) or NR2B selective (threo-ifenprodil) N-methyl-D-aspartate (NMDA) receptor antagonists . The sigma ligand haloperidol and the L-type voltage sensitive calcium channel blocker nimodipine were also effective, whereas the effect of the gamma-aminobutyric acid type A (GABA(A)) receptor agonist muscimol was not significant . Furthermore, chronic ethanol treatment potentiated the NMDA induced neurotoxicity and the ability of acute alcohol to inhibit LDH-release in response to NMDA . According to these results, (i) the phenomenon of alcohol-dependence can be observed at the level of neurones and (ii) NMDA receptors seem to play a central role in the development of ethanol dependence and in neurotoxicity induced by alcohol-withdrawal. Chin J Physiol, 2000 Sep 30, 43(3), 131 - 8 A comparison between acute exposures to ethanol and acetaldehyde on neurotoxicity, nitric oxide production and NMDA-induced excitotoxicity in primary cultures of cortical neurons; Wan JY et al.; Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated processes, such as nitric oxide (NO) formation and excitotoxicity . In the present study, we compared the effects of acute ethanol and acetaldehyde on NMDA receptor-mediated excitotoxicity and NO production in primary cultures of rat cortical neurons . The delayed cell death induced by NMDA (300 mM, 25 min) was evaluated by morphological examination and by measuring the release of the cytotoxic indicator, lactate dehydrogenase, in the culture media 24 hours after the NMDA exposure . The accumulation of nitrite, as an index of NO production, was also measured 24 hours after NMDA treatment . NMDA caused a dose-dependent cell death and nitrite accumulation, both effects were blocked by pretreatment of MK-801 (100 microM) . Acute exposure to ethanol (1-1000 mM) or acetaldehyde (0.1-1 mM) for 35 minutes did not affect neuronal viability in the following 24-hr period . However, acute exposure to acetaldehyde (> or =10 mM) was neurotoxic . Neither ethanol nor acetaldehyde changed basal nitrite levels in the culture media . Acute ethanol (50-400 mM, 10 min) given before the NMDA treatment (25 min) resulted in a concentration-dependent suppression of the delayed cell death . The NMDA-induced NO production was, however, not affected by ethanol . Neither the NMDA excitotoxicity nor NO production was affected by acute ethanol given after NMDA treatment . Acute acetaldehyde (0.01-0.5 mM, 10 min) given before or after NMDA treatment had no effect on delayed NMDA neurotoxicity and NO production . Our data suggest that acute exposure to ethanol is not neurotoxic and is even protective against delayed NMDA-excitotoxicity when given before but not after NMDA treatment . Neither NO nor metabolism of ethanol to acetaldehyde is required for ethanol-mediated suppression of NMDA excititoxicity . Acetaldehyde, on the other hand, is toxic by itself at low concentrations (> or =10 mM) . Furthermore, acute exposure to non-toxic concentrations of acetaldehyde could not protect cortical neurons against NMDA-induced excitotoxicity. Cancer Detect Prev, 2000, 24(5), 405 - 14 Antioxidant compounds interfere with the 3; Natarajan M et al.; Antioxidants are often added to culture media as cytoprotective agents . We examined the effects of antioxidants on the results and interpretation of the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability . Without cells, the thiol-containing antioxidant compounds beta-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-acetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue formazan product in a dose-dependent manner . Addition of the compounds L-ascorbic acid and (+)-alpha-tocopherol acid succinate had different effects . In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thiol groups, did not result in the development of blue formazan product . These results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, readily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present . This undescribed reaction can, therefore, significantly influence the results and interpretation of cell-viability experiments. J Lab Clin Med, 2000 Dec, 136(6), 449 - 56 Influence of cigarette smoking on crocidolite-induced ferritin release by human alveolar macrophages; Plautz MW et al.; Alveolar macrophages (AMs) mobilize iron from the surface of iron-containing minerals such as asbestos and synthesize ferritin for intracellular iron storage or secretion . Although the synthesis of iron-free ferritin (apoferritin) provides antioxidant protection, the secretion of iron-containing ferritin by AMs could increase the availability of catalytic iron in the lungs . Cigarette smoking may promote the secretion of ferritin by AMs after iron acquisition from mineral sources, because smokers' AMs are iron loaded . The first objective of this study was to determine whether ferritin secretion/release by AMs after in vitro exposure to crocidolite asbestos is enhanced by cigarette smoking . The second objective was to assess whether exogenous ferritin-bound iron could enhance the toxicity of crocidolite to lung cells in vitro . AMs recovered from nonsmokers (n = 8) or smokers (n = 8) were exposed to crocidolite or titanium dioxide (TiO2)(1 x 10(6) AMs, 50 to 200 microg/mL) for up to 18 hours . AMs exposed to crocidolite but not TiO2 showed increased cell content of iron and ferritin and increased cell supernatant ferritin concentrations . Increases in iron and ferritin content were similar for AMs recovered from smokers and those recovered from nonsmokers; however, increases in supernatant ferritin were >7-fold greater for smokers' AMs than for nonsmokers' AMs (P < .001) . Exposure of A549 cells, a lung cancer-derived cell line, to crocidolite (50 to 200 microg/mL, 18 hours) caused dose-dependent cell death as indicated by lactate dehydrogenase release . The addition of ferritin (> or = 500 mg/mL) but not apoferritin to culture media enhanced crocidolite-induced LDH release (P < .01) . These findings suggest that cigarette smoking and crocidolite exposure have synergistic effects that promote ferritin release by AMs, which could catalyze oxidative injury to other alveolar cells. Nephron, 2000 Dec, 86(4), 467 - 72 C-Type natriuretic peptide inhibits proliferation and monocyte chemoattractant protein-1 secretion in cultured human mesangial cells; Osawa H et al.; BACKGROUND: Mesangial cell proliferation and matrix accumulation are hallmarks of various progressive glomerular diseases . We examined whether C-type natriuretic peptide (CNP) that is known to regulate the proliferation of vascular smooth muscle cells could modulate these pathological processes using human glomerular mesangial cells (GMCs) in culture . METHODS: Proliferation of GMCs cultured with different concentrations of CNP-22 for 48 h was determined by a colorimetric assay using a tetrazolium salt . Monocyte chemoattractant protein-1 (MCP-1) and type IV collagen secretion into the culture media by GMCs in the presence or absence of CNP-22 were evaluated by ELISA . Expression of mRNA for natriuretic peptide receptor B (NPR-B), a specific receptor for CNP, was examined by reverse transcription polymerase chain reaction (RT-PCR) . RESULTS: CNP-22 (1-10 microM) inhibited serum-induced GMC growth in a dose-dependent manner . The amount of MCP-1 in the culture supernatant was increased approximately 2.4-fold by 5 microg/ml of lipopolysaccharide . This increase was inhibited by CNP-22 at 0.1-1 microM in a dose-dependent fashion . CNP-22 (10 microM) inhibited GMC type IV collagen secretion stimulated by 20 ng/ml of platelet-derived growth factor . Expression of NPR-B mRNA was confirmed in GMCs by RT-PCR . CONCLUSIONS: CNP suppresses GMC proliferation and MCP-1 and type IV collagen secretion by GMCs . It may have a therapeutic potential against human proliferative glomerular diseases, especially those with the involvement of monocytes. J Surg Res, 2001 Jan, 95(1), 67 - 72 Shear force regulates matrix metalloproteinase activity in human saphenous vein organ culture; Patterson MA et al.; BACKGROUND: Development of vein graft intimal hyperplasia has been related both to shear force and to the activity of matrix metalloproteinases (MMPs) . Little data are available regarding the effects of shear on MMP expression and activity . The aim of this study was to examine the relationship among shear force, metalloproteinase activity, and intimal thickening in human saphenous vein segments maintained in organ culture . MATERIALS AND METHODS: Segments of human saphenous vein were cultured under static conditions, or perfused under low-flow and high-flow conditions in a perfusion apparatus for 7 days . Metalloproteinase levels and activities were measured using ELISA and substrate gel zymography, respectively . Intimal thickening was determined by morphometric analysis . Results were compared with control vein tissue, which was not subjected to organ culture, using a one-way ANOVA . RESULTS: A 13% increase in proteolytic activity was noted on substrate gel zymography at 68-72 kDa in high-flow vein tissue . The protein content of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 was increased in high-flow vein tissue by 21%, 126%, more than 100-fold, and 86%, respectively . In culture media bathing the outside of the vein, TIMP-2 was increased in high-flow specimens, while TIMP-1 was inversely related to flow rate . Intimal thickening was directly related to flow rates, and was progressively increased in the low-flow and high-flow groups by 3-fold and 4-fold, respectively . CONCLUSIONS: Metalloproteinase levels in human saphenous vein cultures are related to shear force . MMP levels and activity correlate with the degree of intimal thickening . This model may provide a valuable tool for the analysis of physical forces and their influence on intimal thickening in human saphenous vein . Fertil Steril, 2000 Dec, 74(6), 1220 - 6 Expression of vascular endothelial growth factor mRNA in human preimplantation embryos derived from tripronuclear zygotes; Krussel J et al.; OBJECTIVE: To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos . DESIGN: Human preimplantation embryos not suitable for uterine transfer were examined for beta-actin and VEGF mRNA expression . Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion . SETTING: Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Dusseldorf, Germany . PATIENT(S): Couples undergoing IVF by intracytoplasmic sperm injection for various reasons . INTERVENTION(S): Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion . MAIN OUTCOME MEASURE(S): Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA . RESULT(S): VEGF mRNA and protein could not be detected in unfertilized oocytes . However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16-cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts) . The VEGF protein level was below the sensitivity of the ELISA . CONCLUSION(S): Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival. Cancer Res, 2000 Dec 1, 60(23), 6670 - 6 Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production; Zeng G et al.; Ganglioside GD3 is overexpressed in many types of tumors and may be associated with tumor progression and the development of metastatic potential . In our previous study (G . Zeng et al., Biochemistry, 38: 8762-8769, 1999), we established a subclone of the rat dorsal root ganglion-derived F-11 cells in which the expression of ganglioside GD3 was inhibited by stable transfection of the antisense vector against CMP-NeuAc: GM3 alpha2-8 sialyltransferase (GD3-synthase) gene . This cell line exhibits markedly reduced rate of tumor growth in vivo . Here, we further characterized the antisense-transfected cell line, and the results showed that these cells formed small, minimally vascularized tumors exhibiting extensive necrosis . In vivo Matrigel assay revealed reduced vascularization and low hemoglobin content in the antisense xenografts . Significantly fewer new vessels were found on the antisense xenografts and the skin around them than those on/around the xenografts formed by the sense-transfected and untransfected F-11 cells . The hemoglobin content of the antisense xenografts was much lower than that of the xenografts formed by the control cells . The reduced angiogenesis in the antisense xenografts was correlated with a decrease in vascular endothelial growth factor (VEGF) production . The expression of VEGF was suppressed in the antisense xenografts and the conditioned culture media of the antisense-transfected F-11 cells as determined by Western blotting analysis . This was further confirmed by immunohistochemistry of the tumors using antibodies against VEGF and platelet/endothelial cell adhesion molecule (PECAM-1) . Therefore, our results demonstrate that reduced tumor growth in nude mice by suppression of GD3-synthase expression in F-11 cells results from minimal angiogenesis of the tumors through down-regulation of the VEGF expression, which indicates an important role for GD3 in tumor angiogenesis. Diabetes, 2000 Dec, 49(12), 2170 - 7 Defective intracellular antioxidant enzyme production in type 1 diabetic patients with nephropathy; Ceriello A et al.; There is an individual susceptibility to diabetic nephropathy, and oxidative stress is believed to play an important role in the pathogenesis of diabetic complications . Active oxygen species induce antioxidant enzyme expression in tissues, an effect considered to be a defensive mechanism . To test whether altered intracellular antioxidant enzyme production might explain the predisposition to diabetic nephropathy, we studied the effect of long-term (12 weeks) exposure to normal (5 mmol/l) or high (22 mmol/l) glucose concentrations on fibroblast antioxidant enzyme gene expression and protein activity in type 1 diabetic patients with and without nephropathy, nondiabetic nephropathic patients, and nondiabetic control subjects . Under conditions of normal glucose concentration in the culture media, CuZnSuperoxide-dismutase, MnSuperoxide-dismutase, catalase, and glutathione-peroxidase activity and mRNA expression were not different among the four groups . Under high-glucose conditions, CuZnSuperoxide-dismutase mRNA and activity increased similarly in all groups (P < 0.001 vs . basal), whereas MnSuperoxide-dismutase did not change . In contrast, catalase mRNA and activity as well as glutathione-peroxidase mRNA and activity increased in fibroblasts from type 1 diabetic patients without nephropathy (P < 0.001), in fibroblasts from nondiabetic nephropathic patients (P < 0.001), and in fibroblasts from nondiabetic control subjects (P < 0.001), but not in fibroblasts from type 1 diabetic patients with nephropathy . Exposure to high glucose concentrations significantly increased lipid peroxidation in cells, higher levels being found in cells from diabetic patients with nephropathy (P < 0.001) . These data, while confirming that exposure to high glucose concentrations induces an antioxidant defense in skin fibroblasts from normal subjects, demonstrate a failure of this defensive mechanism in cells from type 1 diabetic patients with nephropathy, whereas skin fibroblasts from diabetic patients without complications or from nondiabetic nephropathic patients have an intact antioxidant response to glucose-induced oxidative stress. Indian J Exp Biol, 2000 Jun, 38(6), 593 - 7 Microspore culture in Corchorus olitorius: effect of growth regulators, temperature and sucrose on callus formation; Ali MA et al.; Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C . olitorius . The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo . Initially, 10-30% microspores divided symmetrically, but only 0.2-1% of the dividing microspores continued dividing and produced multicellular microcalli . About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA . Incubation in the dark at temperatures of 35 degrees C for 1 day and then 25 degrees C was found effective for induction of first embryonic division in Corchorus . The frequency of microspore callus formation was higher on medium containing either 3% or 5% sucrose . Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius . On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced. Chest, 2000 Dec, 118(6), 1828 - 9 Intracardiac mass complicating Malassezia furfur fungemia; Schleman KA et al.; Malassezia furfur is a lipophilic yeast known to colonize indwelling catheters . Although progression to vasculitis and sepsis has been described, it has rarely caused fungemia in adults receiving nutrition via an indwelling catheter . Difficulty in diagnosis occurs as M furfur does not grow on routine culture media unless it is supplemented with fatty acids . We present the first case of M furfur fungemia in an adult, complicated by a pedunculated septic thrombus arising from the superior vena cava and extending into the right atrium . Removal of the catheter, amphotericin-B therapy, and surgical debridement were required for cure. Bone, 2000 Dec, 27(6), 785 - 94 Insulin-like growth factor-1 and -2 stimulate osteoprogenitor proliferation and differentiation and adipocyte formation in cell populations derived from adult rat bone; Jia D et al.; In the present study, we test the hypothesis that the stimulation of osteoprogenitor differentiation by the glucocorticoid dexamethasone (Dex) is mediated, at least in part, through components of the insulin-like growth factor (IGF) system . Because it has been suggested that osteoprogenitors and adipocyte progenitors originate from the same precursor cells, and that their differentiation in many systems is reciprocally regulated, the effects of Dex and IGF on adipocyte formation were also evaluated in the same cultures . In view of the presence of IGFs and their binding proteins in serum, we also evaluated to what degree the effects of IGF-1 and IGF-2 on differentiation of osteoblasts and adipocytes were affected by the serum concentration of the culture media . Bone cell populations were isolated from vertebrae of 3-month-old female Wistar rats using an explant culture technique . Osteoprogenitor differentiation was evaluated by a colony assay: Bone-forming osteoblastic colonies (bone nodules) derived from single osteoprogenitors were identified by alkaline phosphatase (ALP) staining and/or staining for mineralized matrix according to the von Kossa technique . Unmineralized nodules and osteoblastic colonies were subsequently identified by their distinctive color and morphology after methylene blue counterstaining . Differentiated adipocytes were identified by Sudan IV staining . IGF-1 and IGF-2 stimulated both osteoprogenitor and adipocyte progenitor differentiation in a dose-dependent pattern . The stimulation of osteoprogenitor differentiation by IGF was not dependent on Dex, but differentiation of adipocytes was . The stimulatory effects of IGF-1 and IGF-2 on osteoprogenitor differentiation were greater in media containing 2.5% fetal bovine serum (FBS) than in media containing 5% or 10% FBS, whereas stimulation of adipocyte formation was greater in media containing 10% FBS . Neutralizing antibody against the type 1 IGF receptor (IGF-1R) partially blocked IGF- and Dex-induced osteoprogenitor differentiation, but did not affect IGF-induced adipocyte formation . This suggests that IGF-stimulated osteoprogenitor differentiation is mediated through IGF-1R, that the stimulation of adipocyte formation by IGF is not, that the stimulatory effects of Dex on osteoprogenitor differentiation are partially mediated through IGF-1R, and that the effects on adipocyte differentiation appear to be mediated through signaling pathways other than the IGF-1R. J Exp Bot, 2000 Nov, 51(352), 1861 - 6 The role of abscisic acid in controlling leaf water loss, survival and growth of micropropagated Tagetes erecta plants when transferred directly to the field; Aguilar ML et al.; Plants of Tagetes erecta L . (marigold) cultivated in vitro in ventilated containers exhibited greater control of leaf water loss and increased survival in the field than plants cultivated in sealed containers . Increased field survival of plants cultivated in ventilated containers was attributed to higher levels of endogenous abscisic acid (ABA) . Therefore, ABA was supplied exogenously to plants in sealed or ventilated containers by adding ABA (10(-6), 10(-5), 10(-4) M) to the in vitro culture media in order to evaluate control of leaf water loss, growth and field survival . The addition of 10(-4) M ABA to the culture media in sealed containers produced plants that had similar control of leaf water loss and were morphologically similar to plants cultivated in ventilated containers without the addition of ABA . Field survival of 10(-4) M ABA plants (75%) was increased compared to plants cultivated in sealed containers without ABA (31%), with survival being closer to that of plants cultivated in ventilated containers (90-100%) . Plants cultivated with 10(-4) M ABA (sealed and ventilated) also exhibited increased plant vigour and leaf area in the field compared to plants cultivated without ABA . The results suggest that the limited field survival and growth of plants cultured in vitro are related to the limited ABA concentrations they accumulate while in vitro . Consequently, conditions that increase the endogenous ABA concentrations of in vitro plants (like ventilation or ABA addition to the medium) would improve the control of leaf water loss, field survival and plant vigour. Biochemistry (Mosc), 2000 Nov, 65(11), 1305 - 9 Isolation and properties of peroxidase produced by the fungus Panus tigrinus; Revin VV et al.; Synthesis of peroxidase and laccase by the fungus Panus tigrinus was significantly stimulated by addition of the lignocellulose substrate to the culture media . Peroxidase was isolated from the culture liquid and some properties of the enzyme were investigated . P . tigrinus peroxidase belongs to a group of extracellular peroxidases similar to the plant type peroxidases. J Cell Sci, 2001 Jan, 114(Pt 1), 199 - 205 Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface; deS Senanayake P et al.; Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance . Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces . Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified . Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides . Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated . Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P<0.0001) . In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar . Confocal microscopy of cultures probed with a hyaluronan-specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures . Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix. Dis Aquat Organ, 2000 Sep 28, 42(3), 215 - 9 Prolonged in vitro cultivation of Ichthyophthirius multifiliis using an EPC cell line as substrate; Nielsen CV et al.; The ciliate Ichthyophthirius multifiliis, which normally requires a fish host to develop from the theront stage to the trophont stage, was cultivated in vitro for part of its life cycle . Experiments were conducted using a laboratory strain of the parasite originally isolated from rainbow trout Oncorhynchus mykiss in a Danish trout farm . Theronts escaping from tomontocysts were kept in water, cell culture media (E-MEM or L-15), or cultures of EPC (Epithelioma Papulosum Cyprini) cells in plastic tissue culture dishes (Nunc multidish plates) . In addition, a 2-compartment system, with water separated from tissue culture media by a monolayer of EPC cells on an Anopore Tissue Culture Insert (mimicking the fish epidermis) was tested as an experimental habitat for the parasite . Theronts transformed into trophonts in all treatments except in water alone . However, development was accelerated in wells containing EPC cells, and survival and growth of trophonts were significantly increased compared to water or tissue culture media alone . Further, the 2-compartment system allowed superior performance of the parasites (attachment of parasites to cells and growth from 36 to 46 microm) . In all experiments it was found that the presence of host factors (mucus and serum) stimulated parasite development. Prostate, 2000 Dec 1, 45(4), 304 - 14 Alterations of prostate biomarker expression and testosterone utilization in human LNCaP prostatic carcinoma cells by garlic-derived S-allylmercaptocysteine; Pinto JT et al.; BACKGROUND: This study determined the effects of S-allylmercaptocysteine (SAMC), a phytoconstituent from garlic, on the expression of androgen-responsive biomarkers, prostate specific antigen (PSA), and prostate specific membrane antigen (PSMA), in human prostatic carcinoma cells (LNCaP) . METHODS: Secretion of PSA was determined as well as the activity of PSMA measured as a function of its ability to hydrolyze poly-gamma-glutamated folate and N-acetylaspartylglutamate (NAAG) . Folate hydrolase capacity was also determined in SAMC-treated cells grown in charcoal stripped fetal calf serum (CS-FCS) . In addition, testosterone disappearance was measured from culture media of SAMC-treated LNCaP and PC-3 cells as well as from cell free lysates . RESULTS: PSA secretions were significantly decreased compared to control values at 1 day (8.4 +/- 2.6 vs . 18.9 +/- 1.7, P < 0.01), 4 days (18.9 +/- 5.3 vs . 73.8 +/- 4 . 4, P < 0.001), and 6 days (35.6 +/- 2.1 vs . 96.5 +/- 17.9 ng/10(5) cells, P < 0.01; mean +/- SD) . By contrast, PSMA activity measured as either folate hydrolase or NAAG dipeptidase (NAALADase) activity increased in cells treated with SAMC . PSMA-folate hydrolase activity in SAMC-treated cells grown in CS-FCS increased beyond that observed in cells grown in CS-FCS alone . Pre-exposure of LNCaP cells to SAMC resulted in enhanced rate of testosterone disappearance from culture media at 6 hr (P < 0.01) and at 48 hr (P < 0.001) compared to media from cells not previously exposed to SAMC . Results similar to these were also observed in androgen-independent PC-3 cells treated with SAMC . In lysates of SAMC-treated LNCaP cells, the rate of testosterone catabolism was twice that from phosphate buffered saline (PBS)-treated cells . SAMC-treated LNCaP cells grown in media supplemented with testosterone temporarily exhibited enhanced growth over a 2 day period but cell numbers declined later to levels similar to those of SAMC treatment . CONCLUSIONS: These results show that SAMC exhibits differential effects on recognized biomarkers for LNCaP cells similar to those produced by androgen deprivation and strongly suggests that this effect may be mediated, in part, by diminishing the trophic effects of testosterone, likely by converting it to metabolites less reactive toward androgen receptors . Exp Eye Res, 2000 Dec, 71(6), 591 - 7 Induction of matrix metalloproteinases 2 and 9 following stress to the lens; Tamiya S et al.; Matrix metalloproteinase 2 and 9 (MMP-2 and 9, also known as gelatinase A and B) have been implicated in a number of eye diseases, but their possible involvement in lens pathology is yet to be determined . In the present study, we therefore investigated a possible role of matrix metalloproteinases in cataract and posterior capsule opacification . Whole porcine lenses were removed from the eye and cultured in either Eagles Minimum Essential Medium (EMEM) or EMEM supplemented with 1 m M hydrogen peroxide . The medium was sampled and changed every 2 days . On some occasions a sham cataract operation was performed on cultured lenses . The resulting capsular bag was secured to a Petri dish and cultured in EMEM . Culture media from all preparations were analysed for MMP-2 and 9 activity by gelatin zymography . Media samples from lenses which maintained clarity over the 6 day culture period did not display any detectable gelatinolytic activity . However, media from cataractous lenses demonstrated a gelatinolytic band, which had similar molecular weights to the pro-form of MMP-2 . In addition to this band, bands with a similar molecular weight to pro-MMP-9 and its dimeric form were also detected in samples obtained from capsular bag preparations within 24 hr . The data presented indicate that normal lenses have undetectable gelatinase activity . However, there is an associated expression of gelatinases with pathological states of the lens, and therefore gelatinase expression could play an important role in cataractogenesis and posterior capsule opacification . Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13949 - 54 Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin; Kane NS et al.; The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid . Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media . The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity . Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin . A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha . To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes . Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels . However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels. FEBS Lett, 2000 Nov 24, 485(2-3), 163 - 7 Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells; Kwon HY et al.; A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of HIV-1 in a bacterial expression vector to produce a genetic in-frame Tat-SOD fusion protein . The expressed and purified Tat-SOD fusion protein in Escherichia coli can enter HeLa cells in a time- and dose-dependent manner when added exogenously in a culture media . Denatured Tat-SOD protein was transduced much more efficiently into cells than were native proteins . Once inside the cells, transduced Tat-SOD protein was enzymatically active and stable for 24 h . The cell viability of HeLa cells treated with paraquat, an intracellular superoxide anion generator, was increased by transduced Tat-SOD . These lines of results suggest that the transduction of Tat-SOD fusion protein may be one of the ways to replenish the Cu,Zn-SOD in the various disorders related to this antioxidant enzyme. Cell Physiol Biochem, 2000, 10(4), 237 - 42 Metallothionein biosynthesis in human RBC precursors; Rahman MT et al.; The in vitro biosynthesis of metallothionein (MT) has been investigated in RBC precursors from human cord blood in order to support the hypothesis for the nucleated precursor origin of MT in human red blood cells (RBC) . Human RBC precursors are obtained by (i) separating glycophorin A(+) (gly A(+)) cells using a magnetic cell sorting (MACS) technique and by (ii) ex vivo expansion of precursors BFU-E (burst forming unit-erythroid) on methylcellulose semi-solid culture media from mononuclear cells of cord blood . Biosynthesis of MT is detected at the protein level, by immuno-histochemical staining using a mouse monoclonal antibody (E9) in ex vivo expanded RBC precursors obtained from BFU-E . Expression of MT is also detected at the mRNA level by MT specific reverse transcriptase polymerase chain reaction (RT-PCR) both in ex vivo expanded precursors from BFU-E and in MACS separated gly A(+) cells . In addition, the expression of the fetal form of MT, MT-0 (also known as MT-1H) at the mRNA level in glycophorin A(+) cells, is also confirmed by cDNA sequencing . With these observations, to our knowledge, MT biosynthesis in human erythroid precursors is reported for the first time . Moreover, the current findings of MT-0 expression at the mRNA level in gly A(+) RBC precursors of hCB has added one more member in the list of cells/organs like fetal liver, human monocytes, non-neoplastic tissues of adenocarcinoma etc., in which the expression of the human fetal form of MT, i.e . MT-0, has also been reported . J Biol Chem, 2001 Feb 16, 276(7), 5101 - 8 Epub 2000 Nov 17. TIP47 associates with lipid droplets; Wolins NE et al.; Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells . TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins . Hence, we hypothesized that TIP47 might be associated with lipid droplets . In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present . When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets . TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates . When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions . Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein. Ann N Y Acad Sci, 2000, 919, 171 - 87 The use of explant lens culture to assess cataractogenic potential; Aleo MD et al.; Explanted cultures of crystalline lenses have been used to investigate mechanisms of xenobiotic-induced cataract formation . However, very few studies have utilized mechanistic information to predict the cataractogenic potential of structurally diverse xenobiotics . The present investigation outlines how visual assessment of lens clarity, biochemical endpoints of toxicity, and mechanisms of lenticular opacity formation can be used to select compounds with a lower probability of causing cataract formation in vivo . The rat lens explant culture system has been used to screen thiazolidinediones against ciglitazone for their direct cataractogenic potential in vitro . The two compounds that were selected as development candidates (englitazone and darglitazone) did not produce cataracts in rats exposed daily for 3 months . The culture system has also been used to illustrate that the lens is capable of metabolizing compounds to reactive intermediates . In this example, the toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a model cataractogen, was attenuated by inhibiting lenticular cysteine conjugate beta-lyase metabolism using aminooxyacetic acid . Finally, this model was used retrospectively to investigate the cataractogenic potential of CJ-12,918 and CJ-13,454 in rats . These compounds showed differences in the incidence of cataract formation in vivo based on differences in hepatic metabolism and penetration of parent drug and metabolites into the lens . The rank order of cataractogenic potential in vitro correlated better with in vivo results when an induced S9 microsomal fraction was added to the culture media . However, the model did not correctly predict the cataractogenic potential of ZD2138, a structurally similar compound . These studies illustrate the use of explant culture to assess mechanisms of cataract formation and outline its use and limitations for predicting cataractogenic potential in vivo. Pharmazie, 2000 Oct, 55(10), 768 - 71 Polysaccharides from Melittis melissophyllum L . herb and callus; Skrzypczak-Pietraszek E et al.; For comparison of the water-soluble polysaccharides from Melittis melissophyllum L . herb with that produced by Melittis callus cultures the polymeric carbohydrates were extracted from both sources, fractionated by IEC and GPC and the respective fractions analysed concerning sugar composition and linkage characteristics . The dominant structures found in all fractions isolated from herb material and callus were type-II arabinogalactans with a (1-->3)-galactose backbone and arabinose-galactose side chains . No significant differences were found between herb and callus polysaccharides . To optimize the Melittis cell culture systems the culture media were varried systematically . A modified Murashige-Skoog (MS) medium, supplemented with GA3, NAA and BAP was found to be most suitable for large-scale production of callus material. Vet Parasitol, 2000 Dec 20, 94(1-2), 117 - 25 In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus; Frazzon AP et al.; Metarhizium anisopliae is a filamentous fungus used for tick control . The in vitro effects of 12 M . anisopliae isolates on engorged Boophilus microplus females were analysed . The most pathogenic isolate (E6S1) caused a 100% death rate when 10(7) spores/ml were used to infect ticks . Isolates of M . anisopliae taken from experimentally infected ticks proved to be more pathogenic than fungus maintained on culture media . A comparison between dsRNA mycovirus-free and infected M . anisopliae isolates suggested that, in general, virus free isolates were more infective . The results showed that the biological control of B . microplus by M . anisopliae infection might constitute an additional method to integrated tick control management. J Cardiovasc Pharmacol, 2000 Nov, 36(5 Suppl 1), S274 - 7 Stretch-induced endothelin-1 production by astrocytes; Ostrow LW et al.; Astrocytes proliferate during central nervous system (CNS) development and then remain quiescent . However, at a site of brain injury, astrocytes re-enter the cell cycle and undergo complex biochemical/functional changes known as reactive gliosis . Gliosis is the most important histopathologic indicator of CNS injury, regardless of etiology . Endothelins (ETs) have powerful mitogenic effects on astrocytes and have recently been implicated in the induction of gliosis . Reactive astrocytes produce, store . secrete and bind endothelin-1 (ET-1) . The stimuli responsible for activating ET production in astrocytes are unresolved . Because of the relationship between stretch and ET production in other cell types, and the observation that ET-1-positive reactive astrocytes appear in mechanically deformed regions, we are examining whether mechanical deformation affects ET-1 production . We expose mature rat astrocyte cultures to mechanical stress using flexible-bottomed culture plates . Mechanical stretch of quiescent, confluent cultures causes an increase in cytoplasmic Ca2+ and inositol trisphosphate (IP3), and a substantial increase in ET-1 production and secretion into the culture media. Cell Mol Biol (Noisy-le-grand), 2000 Nov, 46(7), 1173 - 82 Cell-associated insulin-like growth factor-binding proteins inhibit insulin-like growth factor-I-induced endometrial cancer cell proliferation; Bermont L et al.; Insulin-like growth factor I (IGF-I) is a peptidic growth factor implicated in the proliferation of a wide variety of cell types, and especially endometrial epithelial cells . Its action is modulated by the presence of IGF-binding proteins (IGFBPs) which are secreted by IGF-I target cells . The partition of IGFBPs between cell-associated and soluble form determines the potentiation or the inhibition of IGF-I action . It is commonly accepted that cell-associated IGFBPs potentiate the IGF-I action while the soluble form of IGFBPs has an inhibitory effect . In endometrial adenocarcinoma, IGF-I is involved in tumoral progression and IGFBPs may be key modulators of the IGF-I-induced cell proliferation . Here we showed that the responsiveness of human endometrial adenocarcinoma cells (HEC-IA cell line) to the mitogenic activity of IGF-I was dependent on the pre-incubation conditions . This responsiveness to IGF-I was conditioned by a differential expression of the IGF system components (IGFBPs and IGF-I receptor) and particularly of the IGFBPs . Indeed, the IGF-I-induced proliferation of the HEC-1A cells was attenuated by the presence of cell-associated IGFBPs . Moreover, the IGF-I incubation induced a release of IGFBP-3 in the culture media as the consequence of an interaction between IGF-I and the cell-associated IGFBP-3 . This effect was dose-dependent and was associated with the attenuation of the IGF-I action on cellular proliferation . Thus, IGFBP-3 might be initially expressed as a cell-associated form and then released in the interstitial fluid after a direct interaction with IGF-I . Therefore, in HEC-IA endometrial adenocarcinoma cells responsive to IGF-I, the IGFBP-3 is the main binding protein expressed and both soluble and cell-associated forms act as inhibitors of IGF-I-induced cellular proliferation. Endocr Regul, 2000 Sep, 34(3), 151 - 5 Is thyroid hormone a modulator of estrogen receptor in porcine follicular cells? Gregoraszczuk EL. OBJECTIVE: To examine whether the action of triiodothyronine on aromatase activity in porcine follicular cells is related to the modulation of estradiol receptor . METHODS: Medium and large preovulatory follicles were incubated in Parker medium (M199) supplemented with 5 % of calf serum as a control medium or with addition of triiodothyronine (T3; 10 -9 M), tamoxifen (TMX; 0.1 mM ) or T3+TMX . The media were collected after 48 h, and assayed for progesterone (P4) and estradiol (E2) secretion by RIA .