Culture medium

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 . RESULTS: T3 added to the medium decreased E2 secretion by both medium and large preovulatory follicles (119.7 % and 123.8 %, respectively; P<0.05) . In contrast, T3 increased the secretion of P4 by medium (136 %; P<0 . 05), while decreased the P4 secretion by large preovulatory follicles (123 %; P<0.05) . The effect of TMX added alone was also dependent on follicular development . Estradiol secretion by medium follicles was 2.5 fold higher (p<0.01) than in control and 2.9 fold higher (P<0.01) than in T3 treated cells . In preovulatory follicles basal E2 secretion was not affected by TMX, while 1.2 fold higher (P<0.05) secretion compared to T3 treated cells was noted . On the other hand, TMX suppressed basal P4 secretion in medium and preovulatory follicles 1.5 fold (P<0.01) and 1.3 fold (P<0.05), respectively . The same phenomenon was observed in T3 treated cells . TMX added to the culture media decreased P4 secretion by medium follicles 1.8 fold (P<0.01) and that by preovulatory follicles 1.3 fold (P<0.05) . CONCLUSIONS: The reversed T3 action on estradiol secretion by both medium (P<0.05) and large preovulatory (P<0.01) follicles in TMX treated follicles suggests the up-regulation of ER by triidothyronine.

Toxicology, 2000 Oct 26, 151(1-3), 117 - 26
Effect of a carotene concentrate on the growth of human breast cancer cells and pS2 gene expression; Nesaretnam K et al.; Breast cancer is the most common cancer in women worldwide . The growth of breast cancer cells is either hormone-dependent or hormone-independent . Both types are represented in vitro by the estrogen-receptor positive (ER+) MCF-7 and the estrogen-receptor negative (ER-) MDA-MB-231 cell lines, respectively . The pS2 gene is an estrogen-regulated gene and serves as a marker for the ER+ tumours . Carotenoids are pigments with anti-cancer properties besides having pro-vitamin A, antioxidant and free-radical quenching effects . This study was designed firstly, to compare the effect of palm oil carotene concentrate with retinoic acid on the growth of the ER+ MCF-7 and the ER- MDA-MB-231 cells; and secondly to evaluate the effect of the palm oil carotene concentrate on the regulation of pS2 mRNA . The growth experiments were performed with monolayer cells seeded in phenol red free RPMI 1640 culture media and subsequently treated with varying concentrations of either retinoic acid or palm oil carotenoids . The cell numbers were determined at the start of each experiment and then at successive time intervals . The results showed that the palm oil carotene concentrate caused dose-dependent inhibition of estradiol-stimulated growth of MCF-7 cells but did not affect the proliferation of MDA-MB-231 cells . Retinoic acid caused similar, albeit more potent effects, as significant inhibition was observed at lower concentrations than the palm oil carotenoids . In the pS2 gene expression experiment, cell monolayers were treated with the carotene concentrate (10(-6) M), either with or without supplemented estradiol (10(-8) M), and subsequently the RNA was extracted . Northern blotting was performed and the regulation of pS2 mRNA determined using a 32P-labelled pS2 cDNA probe . The results showed that the palm oil carotene concentrate did not affect the expression of pS2 mRNA and are therefore independent of the estrogen-regulated pathway.

Eur J Ophthalmol, 2000 Jul-Sep, 10(3), 215 - 26
Matrix metalloproteinase 2: involvement in keratoconus; Smith VA et al.; PURPOSE: The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted . An ocular example is keratoconus . The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas . METHODS: The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography . Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents . RESULTS: Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000 . From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2 . Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes . Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes . This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen . CONCLUSIONS: The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2 . Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.

Cancer, 2000 Nov 1, 89(9), 1966 - 75
Mechanism for bone invasion of oral cancer cells mediated by interleukin-6 in vitro and in vivo; Okamoto M et al.; BACKGROUND: Osteoclastic bone resorption is an important step in bone invasion in several malignancies . Although interleukin (IL)-6 accelerates osteoclastic bone resorption, it remains unclear whether IL-6 may be involved in bone invasion of oral cancer . METHODS: The pit formation assay with calf femur-derived bone slices was performed to examine the bone-resorbing activity of osteoclasts and cancer cells . The chemotaxis activity of the culture media was analyzed by the use of Boyden chamber technique . Nude mice, which were inoculated with IL-6-producing oral cancer cells into masseter, were treated with anti-IL-6 neutralizing antibody, and mandibular-bone invasion of the cells was assessed . RESULTS: BHY, a bone-invasive oral cancer cell line, but not HNT, a noninvasive cell line, produced large amounts of IL-6 . In a pit formation assay, addition of conditioned medium (CM) derived from BHY but not HNT increased osteoclastic bone resorption, and the effects were inhibited by anti-IL-6 antibody . BHY-secreted IL-6 showed significant chemotaxis activity for osteoclasts . Of note, CM from the cocultivation of osteoclasts and BHY markedly enhanced the cancer cell migration, and the chemotaxis activity was significantly reduced when anti-IL-6 antibody was added into the coculture and then CM were collected, but not when the antibody was added into the CM after they were collected . Furthermore, treatment with anti-IL-6 antibody almost completely inhibited mandibular bone invasion of BHY in nude mice . CONCLUSIONS: These results strongly suggest that IL-6 secreted by oral cancer cells plays a significant role in bone invasion .

J Periodontol, 2000 Oct, 71(10), 1575 - 82
Cyclooxygenase-2-dependent prostaglandin production by peripheral blood monocytes stimulated with lipopolysaccharides isolated from periodontopathogenic bacteria; Noguchi K et al.; BACKGROUND: Prostaglandin E2 (PGE2) plays important roles in the pathogenesis of periodontal disease . Recent studies have revealed the existence of 2 isozymes of cyclooxygenase (COX), called COX-1 and COX-2 . The purpose of the present study was to investigate the contribution of COX-1 and COX-2 to PGE2 production by human peripheral blood monocytes that are stimulated with lipopolysaccharides (LPS) from periodontopathogenic bacteria . METHODS: LPS were isolated from Actinobacillus actinomycetemcomitans (A . actinomycetemcomitans) and Porphyromonas gingivalis (P . gingivalis) by the phenol-water method . Peripheral blood monocytes were stimulated with LPS for the indicated periods, and the levels of PGE2 or interleukin (IL)-1 beta in the culture media were measured by enzyme-linked immunosorbent assay . Expression of COX-1 and -2 proteins was studied by immunocytochemical staining, and COX-2 mRNA expression was examined by Northern blot analysis . RESULTS: Peripheral blood monocytes stimulated with A . actinomycetemcomitans- or P . gingivalis-LPS produced PGE2 in a time- and dose-dependent manner . Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a specific COX-2 inhibitor, completely inhibited PGE2 production . Immunocytochemical staining of COX-1 and COX-2 proteins showed that expression of COX-2 protein was increased in monocytes that were stimulated with A . actinomycetemcomitans- or P . gingivalis-LPS, compared with that in unstimulated monocytes, whereas expression of COX-1 protein was not altered . Northern blot analysis showed that monocytes stimulated with A . actinomycetemcomitans- or P . gingivalis-LPS expressed COX-2 mRNA, while COX-2 mRNA was not detectable in unstimulated cells . Treatment of A . actinomycetemcomitans-LPS-stimulated monocytes with NS-398 induced a significant increase of IL-1 beta production to the same extent as treatment with indomethacin . CONCLUSIONS: These results suggest that COX-2 is induced in monocytes stimulated with LPS derived from A . actinomycetemcomitans and P . gingivalis and that the COX-2 is primarily responsible for PGE2 production . COX-2 may be pivotal in PGE2 production in periodontal lesions and may be involved in inflammatory responses.

Connect Tissue Res, 1998, 39(4), 233 - 44
COMP (cartilage oligomeric matrix protein) is synthesized in ligament, tendon, meniscus, and articular cartilage; Muller G et al.; The presence of cartilage oligomeric matrix protein (COMP) in extracts of ligament, tendon, meniscus, and canine articular cartilage was demonstrated by Western blot analysis using anti-dog COMP antibody . When the tissues were cultured in the presence of {35-S}methionine/cysteine, metabolically labeled COMP was purified from the culture media and from tissue extracts by DEAE-cellulose gel chromatography . SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography and immunoblotting under reducing and non-reducing conditions revealed that COMP is synthesized by the cells of these connective tissues . Increased levels of COMP in samples of both synovial fluid and serum of patients with various joint diseases may not only be derived from cartilage but also from ligaments and tendons . COMP is not a highly tissue-specific cartilage molecule.

Carcinogenesis, 2000 Nov, 21(11), 1947 - 57
SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells; Spink BC et al.; In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin . Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1 . In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase . In this study, conjugation of these estrogen metabolites was investigated . A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated . Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry . To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction . Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines . Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells . Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity . The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate . These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

Appl Environ Microbiol, 2000 Nov, 66(11), 5024 - 9
Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of the hemolysin system; Fernandez LA et al.; A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants of Escherichia coli cultures is reported . The method is based on the well-characterized hemolysin transport system (Hly) of E . coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate . The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification . In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

Invest Ophthalmol Vis Sci, 2000 Nov, 41(12), 3833 - 41
Human trabecular meshwork cells secrete neurotrophins and express neurotrophin receptors (Trk); Wordinger RJ et al.; PURPOSE: The purpose of this study was to compare the mRNA expression of neurotrophins (NTs) and NT receptors (Trk) in cultured human trabecular meshwork (HTM) cells and ex vivo HTM tissues, to immunolocalize both NT and Trk receptors in cultured HTM cells, and to demonstrate secretion of NTs by HTM cells . METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of NT and Trk receptor mRNAs in early-passaged, cultured HTM cells from donors of several ages . RT-PCR was used on ex vivo HTM tissues from donors to compare and contrast mRNA expression with cell culture results . In addition, immunohistochemistry was used to localize the translated NT and low- (p75) and high- (Trk) affinity NT receptor proteins within cultured HTM cells and trabecular meshwork tissues . Last, enzyme-linked immunoassay (ELISA) was used to demonstrate secretion of NTs by HTM cells . RESULTS: Amplification products of the expected size for NTs were detected in both cultured HTM cells and ex vivo HTM tissues . Specifically identified were amplification products for the following NTs: NGF, BDNF, NT-3, and NT-4 . Amplification products for the full-length Trk A and Trk C high-affinity receptor were observed, as well as truncated isoforms for Trk B and Trk C . No amplification products were produced for the full-length Trk B receptor nor for the low-affinity p75 receptor . Immunohistochemistry indicated that proteins for the various NTs and full-length and truncated Trk receptors were translated by cultured HTM cells and tissues . Immunoassays (ELISA) detected BDNF, NT-4, NGF, and NT-3 in the culture media from HTM cells . CONCLUSIONS: The results demonstrate, for the first time, mRNA expression for NT and Trk receptors by both cultured HTM cells and ex vivo HTM tissues . NTs were immunolocalized in HTM tissues and cultured HTM cells are capable of secreting NTs . Specific NTs acting through high-affinity Trk receptors within the HTM may be involved in maintaining the normal function of this complex tissue.

Biol Trace Elem Res, 2000 Summer, 75(1-3), 235 - 44
Effects of uranium poisoning on cultured preimplantation embryos; Kundt M et al.; The toxic effect of uranium in cultured preimplantation embryos of the mouse is presented . Embryos were obtained from hybrid females CBA x C57 BL following induction of superovulation and were incubated in M16 cultured medium . Two different experiments were performed . In one, embryos in a one-cell stage were placed in culture media with final concentrations of uranyl nitrate of 104 and 208 microg/mL during 120 h in the same dish . In the other experiment, embryos in a one-cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104, and 208 microg/mL . At 24 h, those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions with uranyl nitrate were added . The percentage of embryos in two-cell stage, morula, early blastocyst, expanded blastocyst, and hatched blastocyst were recorded at 24, 72, 96 and 120 h of culture . The results obtained showed that concentrations as from 26 microg U/mL induced the delay of embryo development and the impairment of blastomere proliferation . The toxic effect of uranium increased in those experiments in which the embryos were transferred to a new medium . This embryo-culture system appears to be appropriate to evaluate the toxic effect of uranium on embryos removed from maternal influences and represents a suitable test system for environmental pollutants.

Blood, 2000 Nov 1, 96(9), 3181 - 7
In vitro and in vivo production of vascular endothelial growth factor by chronic lymphocytic leukemia cells; Chen H et al.; Expansion of primary solid tumors and their malignant dissemination are angiogenesis-dependent . Vascular endothelial growth factor (VEGF) is the key factor playing a pivotal role in solid tumor-induced angiogenesis . Recent studies indicate that angiogenesis may also be involved in the pathogenesis of certain hemic malignancies, including B-cell chronic lymphocytic leukemia (B-CLL) . Mechanisms underlying angiogenesis in B-CLL and the role of VEGF in this process are incompletely understood . In this study, it was examined whether angiogenically functional VEGF is produced by B-CLL cells . Immunohistochemical staining with antibodies against VEGF and CD34, an endothelial cell marker, demonstrated the presence of VEGF protein and abundant blood vessels in infiltrated lymphoreticular tissues . Low levels of VEGF were detected by ELISA in the culture media of unstimulated cells; this was enhanced up to 7-fold by hypoxic stimulation . SDS-PAGE and Western blot analysis of the concentrated culture media showed 2 isoforms of VEGF protein with molecular weights of 28 and 42 kd, respectively . RNA hybridization showed that these cells expressed VEGF mRNA . Reverse transcription-polymerase chain reaction, combined with nucleotide sequence analysis, revealed that the predominantly expressed isoforms were VEGF121 and VEGF165 . Moreover, (3)H-thymidine incorporation and an in vivo angiogenic assay demonstrated that the VEGF produced by CLL cells can induce angiogenesis by stimulating endothelial cell proliferation . In conclusion, this study shows that B-CLL cells produce VEGF and demonstrates the angiogenic effects of this growth factor, which may be relevant for the tissue phase of the disease.

Mol Hum Reprod, 2000 Nov, 6(11), 1033 - 40
Prostaglandin E(2)-dependent production of latent matrix metalloproteinase-9 in cultures of human fetal membranes; McLaren J et al.; Studies in our laboratory have shown that structural changes in cervical biopsied fetal membranes, prior to labour, coincide with differences in the expression of the gelatinase enzyme, latent matrix metalloproteinase-9 (MMP-9) . Concurrently, in vivo, there is an increase in the expression of prostaglandins, notably prostaglandin E(2) (PGE(2)), which has been shown to regulate the expression of MMPs in other systems . The aim of this study was to test the hypothesis (using an in-vitro culture model) that endogenously produced PGE(2) has a role in the elevation of MMP-9 described in vivo . Non-infected fetal membranes sampled from women undergoing elective Caesarean section were stimulated with 10% (v/v) fetal bovine serum (FBS), a known inducer of prostaglandins . This activation resulted in a time-dependent increase in the secretion of PGE(2) into the media, as determined by enzyme-linked immunosorbent assay (day 1: 19 +/- 9 pg/ml/24 h to 358 +/- 54 pg/ml/24 h by day 4) . A similar pattern of secretion of latent MMP-9 was observed in parallel with the increase in PGE(2) in the same culture media (day 1: 1.63 +/- 0.17 ng/ml/24 h to 4.2 +/- 1.4 ng/ml/24 h by day 4) . When both molecules were compared, a significant (P: < 0.01) positive correlation (r = 0.623) was observed . Secretion of the tissue inhibitor of MMPs-9 (TIMP-1) was not significantly different between untreated (3.07 +/- 0.266 microg/ml/24 h) and FBS-treated (3 . 85 +/- 0.24 microg/ml/24 h) cultures during the first 4 days in culture . Prostaglandin synthesis inhibition studies using indomethacin (100 micromol/l) resulted in a 70-80% reduction in the activated secretion of latent MMP-9 . Direct PGE(2) stimulation of cultures resulted in the bell shaped dose-response curve with concentrations of 1-100 nmol/l (which are within the range secreted in culture in response to FBS), stimulating significant latent MMP-9 secretion . These results suggest a link between endogenous PGE(2) and latent MMP-9 production in human fetal membranes, raising the possibility that PGE(2) has a role in the mechanism of fetal membrane structural changes and, hence, in parturition-associated membrane rupture.

Pflugers Arch, 2000 Oct, 440(6), 908 - 17
Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3-absorption to norepinephrine; Kunimi M et al.; Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE) . Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640) . Compared to G-Ringer sol., application of LAG-Ringer sol . in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance . However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55% . While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol . it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100% . This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors . Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP . We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.

Hum Reprod, 2000 Jul, 15 Suppl 2, 199 - 206
Toxic effects of oxygen on human embryo development; Catt JW et al.; The toxic effects of oxygen on the embryos of various animal species are reviewed . Methodologies for assessing embryonic damage are discussed and possible ways of preventing the damage are explored . Three methods of potentially minimizing oxidative damage to human embryos were tested using gametes, zygotes, and embryos from a clinical IVF programme: (i) decreasing the oxygen tension in the gas phase used for culture during insemination, fertilization, and embryo growth; (ii) changing the formulation of culture media to include some components designed to protect against oxidative damage; and (iii) reducing the duration of insemination to minimize the effect of oxidative damage caused by spermatozoal metabolism . Fertilization, cleavage, embryo utilization, pregnancy, and embryo implantation rates were used to monitor these changes . Although all three methods gave an increase in success rates, there was still a dramatic decrease in success with patient age . It is suggested that, although the system of handling and culturing embryos can be optimized with respect to embryonic mitochondrial function, there are inherent age-related defects in oocytes and embryos that are still more fundamental than the environmental conditions of the embryo.

Arthritis Rheum, 2000 Oct, 43(10), 2152 - 9
Acidic fibroblast growth factor in synovial cells; Thomas JW et al.; OBJECTIVE: To characterize the production and regulation of acidic fibroblast growth factor (aFGF) in type B (fibroblast-like) synoviocytes cultured from both inflammatory and noninflammatory synovial lesions . METHODS: Immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction were used to examine the expression of aFGF by synovial cells in vitro . Incorporation of 3H-thymidine by NIH3T3 cells in the presence or absence of neutralizing antibody to aFGF was used to measure bioactive aFGF levels in culture media . RESULTS: Acidic FGF was detected in all synovial cell lines during growth in vitro; however, synoviocytes from rheumatoid arthritis (RA) patients sustained more abundant production of cytoplasmic and nuclear aFGF . Acidic FGF production persisted after multiple passages and did not depend on the presence of serum . Both RA and noninflammatory synovial cells were competent to release aFGF into the media, even though aFGF lacks a signal peptide . Tumor necrosis factor alpha, interleukin-6, and epidermal growth factor did not increase aFGF expression in vitro; in contrast, transforming growth factor beta1 (TGFbeta1) was found to markedly increase aFGF production by cultured synovial cells . CONCLUSION: Acidic FGF synthesis and release is a component of synovial cell growth that is markedly increased in RA . TGFbeta1, and not proinflammatory cytokines, is a potent inducer of aFGF production by synoviocytes in vitro . These findings suggest that in RA, interactions between TGFbeta1 and aFGF may contribute to angiogenesis and fibroblast proliferation, potentially independently of inflammatory mediators.

J Biotechnol, 2001 Nov 17, 84(1), 45 - 52
Real time monitoring biomass concentration in Streptomyces clavuligerus cultivations with industrial media using a capacitance probe; Neves AA et al.; On-line monitoring biomass concentration in mycelial fed-batch cultivations of Streptomyces clavuligerus grown with soluble and partially insoluble complex media, was investigated with an in-situ capacitance probe fitted to an industrial pilot-plant tank . Standard off-line and on-line biomass determinations, including cell dry weight, packed mycelial volume, viscosity, DNA concentration and total CO(2) evolution in the exhaust gases, were performed throughout the experiments and compared to on-line capacitance measurements . Linear relations between capacitance and all other measurements were developed for both media that hold only in defined process phases, depending on the biomass state and the amount of insoluble matter present . For the industrial complex culture media good linear relations were obtained in the fast growth phase between capacitance and DNA concentration and total CO(2) evolution, while in the subsequent transition and stationary phases only with apparent viscosity was a reasonable correlation found . The capacitance probe was shown to be a valuable tool for real-time monitoring biomass concentration in industrial-like cultivation of mycelial streptomycetes.

Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 455 - 61
Phosphatidylinositol 3-kinase signaling to Akt mediates survival in isolated canine islets of Langerhans; Aikin R et al.; The isolation of islet cells from the pancreas by enzymatic digestion causes many of these cells to undergo apoptosis . The aim of this work was to investigate the role of phosphatidylinositol 3-kinase (PI3-K)/Akt signaling in mediating the survival of isolated islets . Insulin-like growth factor-1 (IGF-I) was examined as a potential culture media supplement that could rescue isolated islets from their apoptotic fate . Western blot analysis demonstrated that Akt phosphorylation peaks 20 h after routine islet isolation . PI3-K inhibition with wortmannin abolished both basal and IGF-I-mediated Akt phosphorylation . IGF-I did not increase survival of isolated islets under normal conditions but it did have a protective effect against cytokine (TNF-alpha, IL-1beta, INF-gamma)-mediated cell death . The protective effect of IGF-I against cytokine-stimulated apoptosis was blocked by wortmannin . In addition, inhibition of basal levels of PI3-K activity caused a 31% decrease in islet survival, as shown by MTT assay . These results demonstrate that the PI3-K/Akt pathway mediates survival of isolated islets of Langerhans .

Biochem Biophys Res Commun, 2000 Oct 22, 277(2), 386 - 93
Formic acid dissolves aggregates of an N-terminal huntingtin fragment containing an expanded polyglutamine tract: applying to quantification of protein components of the aggregates; Hazeki N et al.; Huntington's disease (HD) is caused by an expansion of the CAG repeat that encodes polyglutamine in huntingtin . Transient expression of an N-terminal huntingtin fragment containing an expanded polyglutamine tract induced formation of protein aggregates in cultured cells . The turnover of protein components in such aggregates has been difficult to study because of their insolubility in aqueous solutions . Here we describe a method of solubilizing the aggregates and quantifying their protein components . Insoluble pellets were collected from COS7 cells expressing an N-terminal huntingtin fragment containing an expanded polyglutamine tract and subjected to treatment with various detergent, acid, and alkaline reagents . Treatment with 100% formic acid at 37 degrees C for 30 min induced essentially complete dissociation of the aggregates to monomer . We used this solubilization technique to quantify huntingtin fusion protein in the aggregates formed in transient expression experiments . The frequency of aggregate formation increased when the proteasome inhibitor beta-lactone was added to culture media . However, the total amount of accumulated huntingtin fusion protein did not differ between cells cultured with or without beta-lactone . These results suggest that other protein components which are degraded by the proteasome, in addition to huntingtin, might be related to the dynamics of polyglutamine protein aggregates .

Arterioscler Thromb Vasc Biol, 2000 Oct, 20(10), 2212 - 9
Proliferative effect of lipoprotein lipase on human vascular smooth muscle cells; Mamputu JC et al.; Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions . Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects . The aim of the present study was to examine the effect of LPL on VSMC proliferation . Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth . Addition of VLDLs to the culture media did not further enhance the LPL effect . Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation . Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth . Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation . In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed . Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation . Taken together, these data indicate that LPL stimulates VSMC proliferation . LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect . The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.

Exp Hematol, 2000 Oct, 28(10), 1137 - 46
Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution; Carlens S et al.; OBJECTIVE: In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance . Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation . Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum . While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch . Cell expansion in serum-free media would therefore be preferable . MATERIALS AND METHODS: We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%) . We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets . Buffy-coats from four healthy donors were expanded for 21 days . The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%) . For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors . RESULTS: In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117) . For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance) . When serum was added, this discrepancy was not seen . Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture . Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions . Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures . Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells . CONCLUSION: Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells . These cells can easily be transduced and selected . There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days . The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.

Mol Cell Endocrinol, 2000 Jun, 164(1-2), 53 - 8
Differential regulation by FSH and IGF-I of extracellular matrix IGFBP-5 in bovine granulosa cells: effect of association with the oocyte; Ingman WV et al.; Inhibition of insulin-like growth factor (IGF)-I induced DNA synthesis in bovine oocyte-cumulus complexes (OCCs) caused by follicle-stimulating hormone (FSH) has been linked to changes in the extracellular matrix which do not occur in mural granulosa cells (MGCs) . We investigated regulation by IGF-I and FSH of secreted and extracellular matrix entrapped IGF-binding proteins . OCCs and MGCs from bovine ovaries were cultured in media supplemented with IGF-I and FSH for 24 h . Culture media and extracellular matrix were analysed for IGF-binding proteins by Western ligand blot and immunoblot and found to contain principally IGFBP-3 and -5 . The combined treatment of IGF-I and FSH increased the concentration of IGFBP-3 in OCC and MGC conditioned media by 4- and 6-fold, respectively . Treatment of OCCs and not MGCs with IGF-I and FSH together increased extracellular matrix IGFBP-5 by 2.5-fold . The differential regulation of extracellular matrix IGFBP-5 in OCCs compared to MGCs suggest involvement of changes in the extracellular matrix brought about by IGF-I and FSH in overall regulation of IGF-I in the ovarian follicle.

J Ethnopharmacol, 2000 Nov, 73(1-2), 31 - 7
Inhibition of excitotoxic neuronal death by methanol extract of Acori graminei rhizoma in cultured rat cortical neurons; Cho J et al.; Acori graminei rhizoma (AGR) are reported to exhibit a number of pharmacological actions in the central nervous system . The effects of the methanol extract of AGR on excitotoxic neuronal death were evaluated in the present study using cultured rat cortical neurons . Based on the phase-contrast microscopic examinations of cultures and lactate dehydrogenase activities measured in the culture media, the glutamate-induced excitotoxicity was significantly inhibited by the extract . The inhibitory action of the extract was more potent and selective for the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity . The AGR extract competed with {3H}MDL 105,519 for the specific binding to the glycine site of the NMDA receptor with the IC(50) value of 164.7 microg/ml . Modulation of the NMDA receptor activity by the extract was determined using {3H}MK-801 binding studies . The reduction of the binding in the presence of the extract indicated the receptor inactivation by AGR . These results demonstrated that the methanol extract of AGR exhibited protective action against excitotoxic neuronal death, and that the neuroprotective action was primarily due to the blockade of NMDA receptor function by the interaction with the glycine binding site of the receptor.

J Neuroimmunol, 2000 Oct 2, 110(1-2), 66 - 75
Evidence for an interferon-related inflammatory reaction in the trisomy 16 mouse brain leading to caspase-1-mediated neuronal apoptosis; Hallam DM et al.; The trisomy of human chromosome 21 (Down syndrome) is the leading genetic cause of learning difficulties in children, and predisposes this population to the early onset of the neurodegeneration of Alzheimer's disease . Down syndrome is associated with increased interferon (IFN) sensitivity resulting in unexpectedly high levels of IFN inducible gene products including Fas, complement factor C3, and neuronal HLA I which could result in a damaging inflammatory reaction in the brain . Consistent with this possibility, we report here that the trisomy 16 mouse fetus has significantly increased whole brain IFN-gamma and Fas receptor immunoreactivity and that cultured whole brain trisomy 16 mouse neurons have increased basal levels of caspase 1 activity and altered homeostasis of intracellular calcium and pH . The trisomic neurons also showed a heightened sensitivity to the increase in both Fas receptor levels and caspase 1 activity we observed when IFN-gamma was added to the neuron culture media . Because of the autoregulatory nature of IFN activity, and the IFN inducing capability of caspase-1-activated cytokine activity, our data argue in favor of the possibility of an interferon-mediated, self-perpetuating, inflammatory response in the trisomy brain that could subserve the loss of neuron viability seen in this trisomy 16 mouse model for Down syndrome.

J Invertebr Pathol, 2000 Oct, 76(3), 164 - 8
Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication; Lynn DE; Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus . The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1) . When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium . Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses . A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH) . Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM . Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM . Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium . Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells . In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM . These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies . The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production . These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.

J Biol Chem, 2001 Jan 5, 276(1), 541 - 50
Co-translational interactions of apoprotein B with the ribosome and translocon during lipoprotein assembly or targeting to the proteasome; Pariyarath R et al.; Hepatic lipoprotein assembly and secretion can be regulated by proteasomal degradation of newly synthesized apoB, especially if lipid synthesis or lipid transfer is low . Our previous studies in HepG2 cells showed that, under these conditions, newly synthesized apoB remains stably associated with the endoplasmic reticulum (ER) membrane (Mitchell, D . M., Zhou, M., Pariyarath, R., Wang, H., Aitchison, J . D., Ginsberg, H . N., and Fisher, E . A . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 14733-14738) . We now show that independent of lipid synthesis, apoB chains that appear full-length are, in fact, incompletely translated polypeptides still engaged by the ribosome and associated with the ER translocon . In the presence of active lipid synthesis and transfer, translation and lipoprotein assembly are completed, and the complexes exit the ER . Upon omitting fatty acids from, or adding a microsomal triglyceride transfer protein inhibitor to, culture media to reduce lipid synthesis or transfer, respectively, apoB was degraded while it remained associated with the ER and complexed with cytosolic hsp70 and proteasomes . Thus, unlike other ER substrates of the proteasome, such as major histocompatibility complex class I molecules, apoB does not fully retrotranslocate to the cytosol before entering the ubiquitin-proteasome pathway . Although, upon immunofluorescence, apoB in proteasome-inhibited cells accumulated in punctate structures similar in appearance to aggresomes (cytosolic structures containing molecules irreversibly lost from the secretory pathway), these apoB molecules could be secreted when lipid synthesis was stimulated . The results suggest a model in which 1) apoB translation does not complete until lipoprotein assembly terminates, and 2) assembly with lipids or entry into the ubiquitin-proteasome pathway occurs while apoB polypeptides remain associated with the translocon and attached to the ribosome.

Biochim Biophys Acta, 2000 Sep 29, 1481(2), 289 - 96
Overproduction of beta-1,6-glucanase in Trichoderma harzianum is controlled by extracellular acidic proteases and pH; Delgado-Jarana J et al.; To produce high amounts of extracellular endo-beta-1,6-glucanase, we overexpressed the gene bgn16.2 from Trichoderma harzianum under the control of the pyruvate kinase gene promoter (pki) of T . reesei . Transcription of bgn16.2 gene increased under most conditions but not extracellular beta-1,6-glucanase levels . Relationship of extracellular BGN16.2 protein and presence of proteases was studied in order to maximize production . After changing the carbon and nitrogen sources and buffering the culture media at different pHs, four major proteases, the acidic ones being pH-regulated, were detected . Overexpression of BGN16.2 at low pH resulted in BGN16.2 degradation, due to the induction of aspartyl proteases and to instability at pH below 3 . Maximal overproduction of BGN16.2 albeit pure was achieved in buffered medium, where pH-induced aspartyl proteases were absent or when some nitrogen sources, such as yeast extract, peptone or casein were substrate for these proteases.

Biometals, 2000 Jun, 13(2), 101 - 11
Differences in the growth inhibition of cultured K-562 cells by selenium, mercury or cadmium in two tissue culture media (RPMI-1640, Ham's F-10); Frisk P et al.; Effects of some metals on the growth of cultured human erythroleukemia K-562 cells were investigated when grown in two different types of media based upon RPMI-1640 or Ham's F-10 . The study on proliferation, using RPMI-1640 supplemented with sodium selenite, selenomethionine, mercuric chloride, methylmercuric chloride and cadmium nitrate showed no inhibition of growth at concentrations of 2.5, 25, 25, 2.5 and 25 microM, while at 75, 250, 50, 5 and 50 microM toxicity was apparent . Selenite at 5-50 microM and selenomethionine at 50-100 microM inhibited the growth . In Ham's F-10 supplemented with the same compounds no inhibition was found at concentrations of 5, 10, 25, 1 and 50 microM, while at 50, 100, 50, 5 and 75 microM toxic effects were noted . Selenite 10 microM and selenomethionine 25-50 microM inhibited the proliferation . Measurements of trace element levels in pellets of K-562 cells grown in RPMI-1640 or Ham's F-10 unveiled higher cell contents of cadmium and selenium in cells grown in RPMI-1640, being consistent with higher concentrations of these elements in that medium . Manganese and mercury concentrations were higher in cells grown in Ham's F-10 correlating with a higher medium concentration of these elements . The growth responses and cellular uptake differed between the metals and the selenocompounds and although extrapolating the results to humans is difficult the selenium exposures were in approximately the same order of magnitude as in human exposures . The compounds could be ranked according to decreasing toxicity as: methylmercuric chloride > mercuric chloride, cadmium nitrate, sodium selenite > selenomethionine.

J Clin Microbiol, 2000 Oct, 38(10), 3872 - 5
Evaluation of different preservation and storage methods for Malassezia spp; Crespo MJ et al.; Freezing at -80 degrees C, lyophilization, preservation in distilled water, and storage in different culture media were performed in order to find a suitable method that allowed a prolonged storage of Malassezia spp . Freezing at -80 degrees C was the only method successful at maintaining all species.

J Reprod Fertil, 2000 Sep, 120(1), 41 - 7
Successful capacitation and homologous fertilization in vitro in Calomys musculinus and Calomys laucha (Rodentia - sigmodontinae); Lasserre A et al.; Small South American rodents of the genus Calomys have been used extensively for virology and ecological research . Previous studies have demonstrated that Calomys musculinus and Calomys laucha have a relatively short oestrous cycle and that superovulation and parthenogenetic activation can be induced . The purpose of this study was to determine the requirements for in vitro manipulation of the male gamete and in vitro fertilization . Two culture media and different concentrations of spermatozoa were tested for their ability to support sperm motility, hyperactivation and the acrosome reaction . The ability of capacitated Calomys spermatozoa to penetrate zona-free hamster eggs was also evaluated . In vitro fertilization was assessed by examining attachment and binding to the zona pellucida, second polar body extrusion, pronucleus formation and the fertilizing sperm tail . The results of the study showed that: (i) Tyrode's albumin lactate pyruvate (TALP) medium was more effective than T6 medium for maintaining sperm motility in vitro; (ii) hyperactivation was achieved with TALP but not with T6; (iii) the acrosome reaction was easily distinguished by light microscopy and depends on time and sperm concentration; (iv) capacitated spermatozoa are able to penetrate zona-free hamster eggs; and (v) superovulated oocytes can be fertilized in vitro . This is the first report of capacitation and in vitro fertilization for Calomys sp . These results provide opportunities to use C . musculinus and C . laucha as new laboratory animals for research into reproductive biology.

Am J Physiol Gastrointest Liver Physiol, 2000 Oct, 279(4), G767 - 74
Separate pathways for cellular uptake of ferric and ferrous iron; Conrad ME et al.; Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated . Neither the ferric nor ferrous pathway was shared with either zinc or copper . Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron . We postulate that ferric iron was transported into cells via beta(3)-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2) . These conclusions were documented by competitive inhibition studies, utilization of a beta(3)-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways . Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.

Histol Histopathol, 2000 Oct, 15(4), 1145 - 50
Melanization stimulating factors in the integument of the Mugil cephalus and Dicertranchus labrax; Zuasti A et al.; The pigment pattern expression resides in the chromatoblasts of the embryonic skin . The differentiation of these chromatoblasts is influenced by specific local factors such a melanization inhibiting factor (MIF) and a melanization-stimulating factor (MSF) . We reveal the presence of these factors by means of a series of experiments on the skin of the marine species of fish Dicertranchus labrax and Mugil cephalus, each with different pigment pattern, the former having a light skin and the latter a darker one . Media conditioned by exposure to dorsal and/or ventral skin, stimulates the melanization of Xenopus laevis neural crest cells throughout a 3 day assay period . Similarly conditioned culture media tested on B16-F10 murine malignant melanocytes, revealed a considerable influence in enzymatic activities: dopachrome tautomerase (DCT), tyrosine hydroxylase and dopa oxidase . The use of media in a dose response basis suggests that the conditioned media may contain both melanophore stimulating and inhibiting factors . The results obtained may actually reflect the resultant activity of the two factors present.

Biochim Biophys Acta, 2000 Jun 21, 1492(1), 285 - 8
Molecular cloning and expression of rat betacellulin cDNA; Tada H et al.; The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney . Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media . Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.

J Clin Pathol, 2000 Aug, 53(8), 615 - 8
Comparison of three isolation systems for the culture of mycobacteria from respiratory and non-respiratory samples; Harris G et al.; AIMS: To compare the recovery of mycobacteria from clinical samples using the MB/BacT rapid culture system with that obtained using egg medium or the Bactec radiometric method . METHODS: The three methods were compared using 681 clinical samples (462 respiratory and 219 non-respiratory samples) and eight external quality control strains . Culture media were incubated at 35-37 degrees C for six weeks in the MB/BacT system and for 12 weeks in the Bactec system and on egg medium . Solid media were examined macroscopically once a week and the Bactec vials were read six times in the first two weeks, and then weekly for the next 10 weeks (a growth index > 50 indicated a positive vial) . The MB/BacT system positive vials were unloaded from the machine as soon as possible after detection . Confirmation of growth for all systems was by Ziehl-Neelson stained smears . Isolates were identified by a combination of phenotypic and molecular methods . RESULTS: Of the 681 clinical samples, 59 (8.7%) were positive on culture, including 23 strains of Mycobacterium tuberculosis . None of the three systems recovered all of the isolates, but each recovered mycobacteria not detected by either of the other two systems . After six weeks incubation, isolation rates were 87%, 78%, and 90%, and mean times to detection were 13, 19, and nine days for the MB/BacT, egg medium, and Bactec systems, respectively . Although the MB/BacT system was slightly slower than the Bactec system, the biomass was greater, allowing earlier use of molecular probes and earlier inoculation of susceptibility tests . CONCLUSIONS: The MB/BacT system provides comparable performance to the Bactec radiometric system, without the problems of disposal of radioactive waste . Optimal recovery is obtained when culture on egg medium is used in conjunction with a rapid culture system.

Oncogene, 2000 Sep 14, 19(39), 4476 - 9
Leukemic cell line, KG-1 has a functional loss of hOGG1 enzyme due to a point mutation and 8-hydroxydeoxyguanosine can kill KG-1; Hyun JW et al.; We tested the cytotoxic action of 8-hydroxyguanine (8ohG) by observing the viability of several leukemic cell lines (KG-1, U937, Jurkat and K 562) in the presence of 8-hydroxydeoxyguanosine (8ohdG), a nucleoside of 8ohG . It was found that 8ohdG showed cytotoxic action only to KG-1 and that only KG-1 showed a homozygous arginine 209 to glutamine mutation in the hOGG1 gene with an almost negligible hOGG1 enzyme activity . Possibly, the selective cytotoxicity in 8ohdG to KG-1 may be due to its low capacity to cope with an increase in the 8ohG level in DNA resulting from the incorporation of 8ohdG present in the culture media . The mutational impairment of hOGG1 in KG-1 is the first report in leukemic cell lines . Using KG-1 with impaired hOGG1, we demonstrated cytotoxicity of 8ohdG probably due to its incorporation into cellular DNA . This new property of KG-1 may allow it to serve as an useful tool for studies of OGG1, oxidative DNA damage and the cytotoxic action of 8ohG . Oncogene (2000) 19, 4476 - 4479.

Int J Impot Res, 2000 Sep, 12 Suppl 3, S25 - 31
Inhibition of Peyronie's plaque fibroblast proliferation by biologic agents; Anderson MS et al.; Peyronie's disease is a fibromatosis of the tunica albuginea which affects up to 2% of men . Plaque development is believed to result, at least in part, from fibroblast proliferation and excess collagen deposition . Numerous oral and intralesional therapies have been used, including verapamil, colchicine and steroids . The purpose of this study was to investigate the in vitro effects of prostaglandin-E1 (PGE1), verapamil and colchicine on the proliferation rates of fibroblasts derived from Peyronie's disease tissue . Using tissue culture, multiple cell lines comprising fibroblasts from Peyronie's plaque, normal tunica and foreskin were established . Cells of low passage were removed from the parent culture and incubated with varying concentrations of PGE1 (0.1-10 mg/ml), verapamil (10-1000 mg/ml), and colchine (2.5 mg/ml) . Proliferation was assessed at 48, 72 and 96 hours using the Vybrant MTT cell proliferation and then compared to control cells . Six plaque lines and 5 normal tunical cell lines were established . These cell lines exhibited excellent linear growth in culture media alone . Co-culture wih PGE1 resulted in no significant inhibition at 0.1 and 1 mg/ml, but a mean inhibition of 60.6+/-11.5% at a concenrtation of 10 mg/ml was noted . Similar inhibition was noted with verapamil at 100 and 1000 mg/ml with a mean inhibition of 65.2+/-10.6% . Colchicine resulted in a mean inhibition of 28% at a concentration of 2.5 mg/ml . Maximum inhibition occurred at 96 hours in all cases . There was no statisitically significant difference in proliferation rates between plaque and normal tunical cell lines . We have developed an in vitro model to assess the effects of biologically active agents on the growth of fibroblasts derived from Peyronie's disease tissue . Our data suggests that PGE1, verapamil, and colchicine inhibit in vitro proliferation of fibroblasts at specific concentrations . Refinement and application of this knowledge may allow the development of useful pharmacologic strategies for men with PD.

J Eukaryot Microbiol, 2000 Sep-Oct, 47(5), 504 - 10
Infection of Gymnodinium sanguineum by the dinoflagellate Amoebophrya sp.: effect of nutrient environment on parasite generation time, reproduction, and infectivity; Yih W et al.; Preliminary attempts to culture Amoebophrya sp., a parasite of Gymnodinium sanguineum from Chesapeake Bay, indicated that success may be influenced by water quality . To explore that possibility, we determined development time, reproductive output, and infectivity of progeny (i.e . dinospores) for Amoebophyra sp . maintained on G . sanguineum grown in four different culture media . The duration of the parasite's intracellular growth phase showed no significant difference among treatments; however, the time required for completion of multiple parasite generations did, with elapsed time to the middle of the third generation being shorter in nutrient-replete media . Parasites of hosts grown in nutrient-replete medium also produced three to four times more dinospores than those infecting hosts under low-nutrient conditions, with mean values of 380 and 130 dinospores/host, respectively . Dinospore production relative to host biovolume also differed, with peak values of 7.4 per 1,000 microm3 host for nutrient-replete medium and 4.8 per 1,000 microm3 host for nutrient-limited medium . Furthermore, dinospores produced by "high-nutrient" parasites had a higher success rate than those formed by "low-nutrient" parasites . Results suggest that Amoebophrya sp . is well adapted to exploit G . sanguineum populations in nutrient-enriched environments.

Vet Surg, 2000 Sep-Oct, 29(5), 420 - 9
Fibrous tissue of subchondral cystic lesions in horses produce local mediators and neutral metalloproteinases and cause bone resorption in vitro; von Rechenberg B et al.; OBJECTIVES: To define the release of nitric oxide (NO), prostaglandin E2 (PGE2), and the neutral metalloproteinases (NMPs) in horses with subchondral cystic lesions (SCL) and to study bone resorption triggered by conditioned media of fibrous tissue of SCL in vitro . STUDY DESIGN: Equine explant cultures of fibrous tissue of SCL, and synovial membrane and articular cartilage of normal horses and horses affected with moderate and severe osteoarthritis were performed . NO, PGE2, and NMP concentrations of media samples were measured, and osteoclast formation and activation was studied in vitro . ANIMALS: Experiment 1: 32 horses with SCL (n = 8), normal joints (7), and joints with moderate (7) and severe (10) osteoarthritis (OA) . Experiment 2: 22 horses with SCL (n = 3), normal joints (7), and chip fractures (12) . Experiment 3: Conditioned media of fibrous tissue from 3 horses with SCL of the medial femoral condyle (n = 1), distal metacarpal bone (1), and tarsal bone (1) . METHODS: Determinations of local mediator concentrations were made with the Griess assay for NO and an enzyme immunoassay kit for PGE2 concentrations in biological fluids . Enzyme activities were assessed with radiolabeled substrates indicating collagenolytic, gelatinolytic, and caseinolytic activities . The resorption pit assay was used to assess osteoclast recruitment and activity . RESULTS: Fibrous tissue of SCL produced NO, PGE2, and NMPs . Of all the variables measured, PGE2 concentrations were the highest in cystic tissue of SCL compared with synovial membrane and articular cartilage from normal joints and joints with chip fractures, indicating that this mediator may play an important role in pathological bone resorption associated with SCL . These findings were supported by the observation that conditioned media of SCL tissue were capable of recruiting osteoclasts and increasing their activity . CONCLUSION: Fibrous tissue of SCL released NO, PGE2, and NMPs into the culture media . It is suspected that intralesional fibrous tissue may play an active role in the pathological process of bone resorption occurring in SCL in horses and may be partly responsible for the maintenance, slow healing rate, and expansion of these lesions . CLINICAL RELEVANCE: Understanding the pathogenesis of SCL will help to establish successful therapy in horses affected with SCL.

Cell Tissue Res, 2000 Sep, 301(3), 353 - 67
Autocrine growth promotion by multiple hematopoietic growth factors in the established renal cell carcinoma line KU-19-20; Tachibana M et al.; Increasing evidence suggests that paraneoplastic syndrome may be mediated by tumor-related cytokine release, although the specific factors involved remain to be clearly defined . The cancer cells used in the present study were obtained from a 67-year-old man with metastatic renal cell carcinoma in the subcutaneous space who demonstrated marked leukocytosis (37,800/mm3) . The primary tumor of the kidney was pathologically diagnosed as renal cell carcinoma consistent with the sarcomatoid type . On microscopic observation, the cultured cells exhibited an epithelial appearance with vacuole formation in their cytoplasm . Ultrastructural observations revealed relatively marked microvilli and a tight junction . Significant amounts of GM-CSF, G-CSF, IL-6, and IL-8 concentrations in the culture media were identified by an enzyme-linked immunosorbent assay . Reverse transcriptase polymerase chain reaction (RT-PCR) significantly exhibited marker protein m-RNA expression in cancer cells . In addition, GM-CSF receptor and IL-6 receptor mRNA expression was also demonstrated by RT-PCR . The administration of both IL-6 and GM-CSF induced cell-proliferation activities estimated by both {3H}-thymidine and bromodeoxyuridine labeling . Anti-IL-6 antibody and anti-GM-CSF antibody neutralized the enhanced proliferative activities generated by these cytokines . Our findings indicate that the established renal cancer cell line can be demonstrated by both the production of multiple cytokines and by their promotion of autocrine growth . These cells are thus considered to be useful as an effective model for multipotent differentiated renal cell carcinoma, as well as for studying the mechanisms of action of autocrine growth.

Acta Ophthalmol (Copenh), 1988 Jun, 66(3), 327 - 33
Corneal graft endothelial cell densities after preservation in M-K, K-sol and MEM tissue culture media . A clinical and specular microscopic study; Ruusuvaara P et al.; The endothelial cell densities of corneal transplants of 30 keratoconus patients have been studied . All operations were performed by the senior author using the same technique . Preservation systems M-K medium, MEM-solution and K-sol were used; ten grafts in each group . The average endothelial cell density for all 30 transplants was 1815 +/- 869 cells/mm2 . The mean cell density was the lowest (1128 +/- 587 cells/mm2) and the mean follow-up time longest (2 years and 9 months) in M-K medium stored grafts . K-sol is the newest preservation method used in our hospital . It gave the best graft endothelial cell densities (2302 +/- 938 cells/mm2) . The mean follow-up time was shortest in K-sol (8 months) . The mean endothelial cell density for grafts from MEM-solution was 2015 +/- 614 cells/mm2) with the mean follow-up time of 1 year, 4 months.

J Biotechnol, 2000 Aug 25, 81(2-3), 129 - 40
Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media; Park H et al.; Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity . To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver . To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells . The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media . The accumulation of ammonium ion was approximately 25-33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines . Interestingly however, the cell growth was approximately 15-30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines . Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.

J Biochem Biophys Methods, 2000 Sep 11, 45(2), 169 - 81
Isolation of thecal cells: an assessment of purity and steroidogenic potential; Li SK et al.; In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined . The estradiol (E(2)) and progesterone (P(4)) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination . The results confirm that no E(2) could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident . The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined . The production of P(4) in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E(2) was detected from the same culture media . In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P(4) (P<0.05) in the serum-free cell culture media . The stimulation of P(4) production was greater at higher LH concentration (50 ng/ml) (P<0.05) . Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05) . The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs . EC(50)>1 ng/ml at LH-50 ng/ml) . The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity . Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production . However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.

Hypertension, 2000 Sep, 36(3), 325 - 9
Fluvastatin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells; Ikeda U et al.; Matrix metalloproteinase-1 (MMP-1), also called interstitial collagenase, may play an important role in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture . We investigated the effects of fluvastatin on MMP-1 expression in human vascular endothelial cells (ECs) . The addition of fluvastatin decreased the basal MMP-1 levels in the culture media of ECs in a time-dependent (0 to 48 hours) and dose-dependent (10(-)(8) to 10(-)(5) mol/L) manner . On the other hand, fluvastatin did not affect tissue inhibitor of metalloproteinase-1 levels . Collagenolytic activity in conditioned media of ECs was also dose-dependently reduced by fluvastatin . The effect of fluvastatin on MMP-1 expression was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene . Inhibition of Rho by C3 exoenzyme also significantly decreased MMP-1 expression in ECs . Our findings revealed that fluvastatin decreases MMP-1 expression in human vascular ECs through inhibition of Rho.

Exp Toxicol Pathol, 2000 Aug, 52(4), 335 - 8
Optimal oxygen tension conditions for viability and functioning of precision-cut liver slices; Drobner C et al.; Optimal oxygenation of culture media is important for the successful use of liver slices as an in vitro tool for studying liver function . For this reason the influence of 20, 40, 70 and 95% O2 concentration on the viability and metabolism of liver slices was investigated . The slices were incubated in the roller system at 37 degrees C under continuous gassing for 2, 24 and 48 hrs . Protein, DNA and potassium contents were maintained or even increased over time without influence by O2 concentrations . The albumin secretion of slices incubated at 40-95% O2 did not differ, but was much lower at 20% O2 . A slight non-significant decrease in albumin secretion after 24 hrs of cultivation could be observed, whereas a much steeper decline was found in all groups after 48 hrs . Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation (ECOD) did not differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation . It can be concluded that O2 concentration of 20% is not sufficient to maintain all cell functions of incubated rat liver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.

Eur J Obstet Gynecol Reprod Biol, 2000 Sep, 92(1), 51 - 6
In vitro development and metabolism of the human embryo up to the blastocyst stage; Devreker F et al.; The preimplantation period begins with the fertilisation of the oocyte and ends with the formation of the blastocyst . During this period, several major events occur resulting in an embryo composed of pluripotent cells . These morphological changes correspond to changes in the embryonic metabolism . The cleavage stages are characterised by a low metabolism and the inability of the embryo to metabolise glucose . After the activation of the embryonic genome, there is a surge in the embryonic metabolism with increased demand for ATP . The embryo is then able to metabolise glucose . Recently, the importance of amino acids has been highlighted by experiments with mouse, hamster and bovine embryos . Amino acids have also been reported to benefit human embryo development in vitro . Some growth factors have been shown to play a role in human embryo development too . The importance of lipids or vitamins, however, is poorly investigated . Culture media have been developed to improve preimplantation development, but more information is required for adapting culture condition to embryonic requirements which hopefully will improve the outcome of in vitro fertilisation (IVF).

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 121 - 6
Enzymes of Botrytis cinerea capable of breaking down hydrogen peroxide; Gil-ad NL et al.; The amounts of intra- and extracellular guaiacol peroxidase, ascorbic peroxidase, glutathione peroxidase, superoxide dismutase, laccase, and catalase present in Botrytis cinerea, cultured in three different media: Kovac synthetic medium, Sabouraud fluid medium, and a medium containing malt extract, were determined . The activity of two enzymes, ascorbic peroxidase and glutathione peroxidase, has not been previously described in B . cinerea . The detected amount of the enzymes showed considerable variability in the three different culture media . The presence of an array of enzymes capable of metabolizing hydrogen peroxide, whose levels are determined by the conditions under which the fungus grows, shows that B . cinerea is well equipped to contend with the occurrence of host-produced active oxygen species.

Biocell, 2000 Aug, 24(2), 107 - 22
Growth factors and embryo development; Teruel M et al.; In this review are cited and discussed the possible roles of growth factors on preimplantation embryo development of different species . In first term, is considered the mRNA detection in early stages of development . The distribution pattern was not uniform for the different peptides evaluated . For some of them, the mRNAs are detected at the oocyte stage and the level declines to the blastocyst stage, which suggests a maternal origin for them . For others, the level increased from 2-4 cells to blastocyst stage . On the other hand, transcripts of growth factor receptors have been detected in preimplantation embryos . This suggests that growth factors of maternal or embryo origin interact with specific receptors on preimplantation embryo surface and regulate the early development . On the other hand, culture media supplemented with different growth factors have been used to study the possible effects on in vitro development . Some investigators have found no effect . Others, however, have demonstrated changes in protein synthesis, cell number, differentiation and hatching processes . Embryo development modulation by growth factors probably involves a balance between stimulatory and inhibitory effects, although works are needed to determine the precise roles played by these polypeptides during early stages of mammalian development.

Hokkaido Igaku Zasshi, 2000 Jul, 75(4), 237 - 42
{Present state and future in reproductive medicine}; Sengoku K; It has been almost 21 years since the first birth of IVF baby and rapid advances in assisted reproductive technology (ART) have taken place . Today, several new technology have been developed such as microfertilization and cryopreservation of embryos and ART has become an important and popular tools for treatment of infertility patients with several causes . However, the take home baby rates have still been low around 15% . To improve the results in ART programs, the improvement of embryo viability and the solution of problems of implantation have been required . Recently, sequential culture media for production of high quality blastocysts have been developed and results have been as good as with co-culture . These culture media are now commercially available . Several authors reported that higher clinical pregnancy rate was achieved and high-order multiple pregnancy can be eliminated with blastocyst transfer . It has been expected that blastocyst transfer would become the means to solve the major problem that ART has faced such as the low take home baby rates and high-order multiple pregnancy.

Extremophiles, 2000 Aug, 4(4), 247 - 52
A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent; Rothe O et al.; The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems . The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes . We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber . When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber . Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media . Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide . H2S was simply added to the headspace of serum bottles by a syringe . The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide . Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure . For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent . The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably.

Am J Respir Cell Mol Biol, 2000 Sep, 23(3), 411 - 8
Irradiation-induced expression of hyaluronan (HA) synthase 2 and hyaluronidase 2 genes in rat lung tissue accompanies active turnover of HA and induction of types I and III collagen gene expression; Li Y et al.; Hyaluronan (HA) is a linear glycosaminoglycan that accumulates in the interstitium of injured lung and inhibits gas exchange between air and blood . In the present study we investigated the molecular mechanisms behind the local turnover of HA during the early phase of irradiation-evoked lung fibrosis in rats . Irradiation with a single dose of 30 Gy to the lower part of the right lung of rats induced an accumulation of HA in bronchoalveolar lavage fluid 6 wk after irradiation, followed by return to almost normal levels at 10 wk after irradiation . This was parallelled with a transient downregulation of HA receptors on alveolar macrophages (AMs); 4 and 6 wk after irradiation the binding of {(3)H}HA to AMs was decreased to about 50% of that of AMs from nonirradiated control rats, returning to almost normal level at 10 wk after irradiation . Analysis of the expression of rat HA synthase (HAS) isoforms (rHAS1, rHAS2, and rHAS3) and rat hyaluronidases (rHYAL1 and rHYAL2) by Northern blotting revealed an upregulation of rHAS2 messenger RNA at 4, 6, and 10 wk after irradiation, but a progressive decrease in the constitutive expression of rHYAL2 at 6 and 10 wk after irradiation; rHAS1 was undetectable, whereas rHAS3 and rHYAL1 were faintly detectable . Although transforming growth factor-beta1 stimulated HA production by normal lung fibroblasts, it inhibited HYAL activity in lysosomes and HYAL activity released into the culture media . Another interesting observation was that HA fragments, which likely result from the action of HYAL, induced expression of types I and III collagen genes . Our results indicate that rHAS2 and rHYAL2 are involved in the turnover of HA during the early phase of lung injury and that rHAS2 and rHYAL2 as well as HA fragments may play important roles in the pathogenesis of lung fibrosis.

PDA J Pharm Sci Technol, 2000 Jul-Aug, 54(4), 332 - 42
On the cause of performance variation of biological indicator used for sterility assurance; Shintani H et al.; Variations in biological indicator (BI) lethality have been reported for several types of commercial BIs . This phenomenon has been observed among different lots of the same species and strain BIs from a single vendor . It has also been reported among BIs from different vendors but of the same species and strain that are intended to challenge the same general type of sterilization process . Although BI variability has been widely reported, the contributing factors to the variation in observed lethality have not been specifically identified . This is because the previous reports overlooked, to some extent, the differences in carrier materials, primary packaging materials, and culture media used in the manufacture of commercial BIs . The differences in lethality attributable to the carrier material, for so called "substrate effects," have been widely reported . For the BI preparation in this experiment, the same carrier material, primary packaging material, and culture medium were used . The only variable was the use of different spore suspensions supplied from different BI manufacturers . The authors found no significant difference in BI performance as measured by BI resistance . BI population may vary depending on the retrieval technique or population variability in a purchased BI suspension . Unlike some previously published studies, there was no indication from our studies that a specific BI manufacturer supplied BIs with either greater resistance or greater population.

Endocrine, 2000 Jun, 12(3), 273 - 8
Regulation of in vitro maturation of stimulus-secretion coupling in fetal rat islet beta-cells; Sjoholm A et al.; We have studied the maturation of a glucose-responsive insulin release from fetal rat islets, and specifically investigated the impact of nutrients, alpha-adrenoceptors, imidazoline receptors, and cyclic adenosine monophosphate (cAMP) . Islets were isolated from 21 -d-old fetal rats and maintained for 7 d in tissue culture at 3.3 or 11.1 mM glucose and various supplements . Culture in the presence of the nonglucidic nutrient alpha-ketoisocaproic acid (KIC), markedly enhanced both basal and stimulated insulin release from islets cultured at either low or high glucose . Additionally, KIC significantly elevated the insulin content of islets maintained in low glucose, whereas it slightly lowered it in islets cultured at high glucose . Culture with phentolamine, an antagonist of alpha-adrenergic and imidazoline receptors, markedly amplified both basal and glucose-stimulated insulin secretion when added with islets cultured in either low or high glucose . By contrast, the pure alpha2-adrenoceptor antagonist benextramine had no such effects . Addition to culture media of a membrane-permeant agonist (Sp-cAMP{S}) or antagonist (Rp-cAMP{S}) of cAMP-dependent protein kinases types I and II failed to influence basal or glucose-responsive insulin secretory rates at either glucose concentration during culture as well as islet insulin content . In conclusion, islet beta-cell differentiation and functional maturation of the stimulus-secretion coupling can be accelerated in vitro in fetal rat pancreatic tissue by nutrient stimulation, and by interference with imidazoline receptors, whereas cAMP seems virtually ineffective in this respect . These effectors may be of regulatory significance in the in vivo development of glucose-sensitive beta-cells.

Exp Clin Endocrinol Diabetes, 2000, 108(4), 299 - 304
2,3,7,8-Tetrachlorodibenzo-p-dioxin alters follicular steroidogenesis in time- and cell-specific manner; Pieklo R et al.; To show the direct effect of TCDD on ovarian steroidogenesis theca (Tc) and granulosa (Gc) cells were cultured separately or as a co-culture (GT) . Cells were cultured in M199 medium supplemented every day with 0.1 nM TCDD or only at the beginning of the culture with 10 nM of TCDD . After 48, 96 and 144 h culture media were collected for steroids content analysis . In Tc cultured alone TCDD caused an increase in E2 production after 48 and 96 h of culture, while exposure of Tc on TCDD for 144 h caused the decrease in both T and E2 secretion . In Gc cultured alone and in GT cultures the increase in E2 secretion was observed only after 48 h, while a long term exposure to TCDD (96 and 144 h) caused a decrease of both P4 and E2 secretion . The results of the present study suggest various, time-dependent mechanisms of TCDD action on ovarian cells.

J Periodontol, 2000 Jul, 71(7), 1100 - 9
Biological effects of cementum and bone extracts on human periodontal fibroblasts; Hou LT et al.; BACKGROUND: Non-collagenous proteins of mineralized tissues play important roles in bone induction during mineralization and in regulating the activity of many types of mesenchymal cells . This study was conducted to determine the effects of acetic acid extracts of bone and cementum on alkaline phosphatase (ALPase) activity and in vitro mineralization of cultured human periodontal fibroblasts (hPF) . METHODS: Alveolar bone and cementum obtained from clinically healthy subjects were extracted by a solution containing 0.5 M acetic acid and enzyme inhibitors . Osteoblastic phenotypes of hPF were assayed by ALPase activity, gene expression of bone marker proteins, and the ability to produce in vitro mineralization in culture media containing 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate, and 10(-7) M dexamethasone . The effects of cementum and bone extracts on the expression of osteoblastic phenotypes in hPF were also determined . RESULTS: Many protein components, varying in molecular weight from 10 to 14 to 120 kDa, were detectable in 10% SDS-PAGE of both cementum and alveolar bone extracts . The hPF cells were found to exhibit a moderate ALPase activity when compared with rat osteosarcoma (ROS) 17/2.8 cells under the same experimental conditions . Gene expression for ALPase, osteocalcin bone sialoprotein, osteopontin, and BMP-7 at mRNA message was detected by RT-PCR in hPF and ROS 17/2.8 cells . The confluent hPF and ROS 17/2.8 cells showed evidence of calcium deposition in the extracellular milieu at 30 and 15 to 30 days' cultures, respectively, under a mineralization medium . The hPF appeared to form mineralized foci with morphological characteristics different from the mineralized nodules produced by ROS 17/2.8 cells . The addition of low concentrations (5 microg/ml) of either cementum or bone extract produced an increase in the size and number of mineralization spots, as well as greater ALPase activity in both hPF and ROS 17/2.8 cultures during the observation periods . CONCLUSIONS: These results suggest that hPF possess certain mineralizing phenotypes, and that acetic acid extracts of bone and cementum contain components capable of stimulating osteogenic differentiation of hPF.

J Vasc Surg, 2000 Sep, 32(3), 575 - 83
Increased matrix metalloproteinase 2 expression in vascular smooth muscle cells cultured from abdominal aortic aneurysms; Crowther M et al.; OBJECTIVES: Recent evidence has implicated matrix metalloproteinase 2 (MMP-2) in the pathogenesis of aneurysms . The aim of this study was to examine MMP-2 production and expression by aortic smooth muscle cells (SMCs) and dermal fibroblasts derived from patients with abdominal aortic aneurysms (AAAs) . METHODS: Aortic SMCs and dermal fibroblasts were cultured from patients with AAAs or from age-matched controls with atherosclerosis . The production of MMP and tissue inhibitor of metalloproteinase into culture media was analyzed with the use of gelatin zymography, Western blotting, and enzyme-linked immunosorbent assay . Gene expression was analyzed with Northern blotting . RESULTS: All cells studied constitutively produced MMP-2 . Aortic SMCs cultured from aneurysmal tissue expressed MMP-2 protein and messenger RNA at a significantly higher level than SMCs from controls (P =.008) . Dermal fibroblasts from patients with AAAs expressed MMP-2 at a similar level to controls . In both cell types, tissue inhibitor of metalloproteinase 2 and membrane type 1-MMP were expressed at similar levels . CONCLUSIONS: These data suggested that the regulation of MMP-2 gene expression was altered in the aortic SMCs of patients with aneurysms, but this finding was not repeated in other mesenchymal tissue.

Int J Obes Relat Metab Disord, 2000 Aug, 24(8), 989 - 96
Increased adiposity in animals due to a human virus; Dhurandhar NV et al.; BACKGROUND: Four animal models of virus-induced obesity including adiposity induced by an avian adenovirus have been described previously . This is the first report of adiposity induced in animals by a human virus . OBJECTIVE: We investigated the adiposity promoting effect of a human adenovirus (Ad-36) in two different animal models . DESIGN: Due to the novel nature of the findings we replicated the experiments using a chicken model three times and a mammal model once . In four separate experiments, chickens and mice were inoculated with human adenovirus Ad-36 . Weight matched groups inoculated with tissue culture media were used as non-infected controls in each experiment . Ad-36 inoculated and uninfected control groups were housed in separate rooms under biosafety level 2 or better containment . The first experiment included an additional weight matched group of chickens that was inoculated with CELO (chick embryo lethal orphan virus), an avian adenovirus . Food intakes and body weights were measured weekly . At the time of sacrifice blood was drawn and visceral fat was carefully separated and weighed . Total body fat was determined by chemical extraction of carcass fat . RESULTS: Animals inoculated with Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides . This syndrome was not seen in chickens inoculated with CELO virus . Sections of the brain and hypothalamus of Ad-36 inoculated animals did not show any overt histopathological changes . Ad-36 DNA could be detected in adipose tissue, but not skeletal muscles of randomly selected animals for as long as 16 weeks after Ad-36 inoculation . CONCLUSIONS: Data from these animal models suggest that the role of viral disease in the etiology of human obesity must be considered.

J Biomed Mater Res, 2000 Nov, 52(2), 395 - 403
The cytotoxicity of corrosion products of nitinol stent wire on cultured smooth muscle cells; Shih CC et al.; Although nitinol is one of most popular materials of intravascular stents, there are still few confirmative biocompatibility data available, especially in vascular smooth muscle cells . In this report, the nitinol wires were corroded in Dulbecco's modified Eagle's medium with constant electrochemical breakdown voltage and the supernatant and precipitates of corrosion products were prepared as culture media . The dose and time effects of different concentrations of corrosion products on the growth and morphology of smooth muscle cells were evaluated with {(3)H}-thymidine uptake ratio and cell cycle sorter . Both the supernatant and precipitate of the corrosive products of nitinol wire were toxic to the primary cultured rat aortic smooth muscle cells . The growth inhibition was correlated well with the increased concentrations of the corrosion products . Although small stimulation was found with released nickel concentration of 0.95 +/- 0.23 ppm, the growth inhibition became significant when the nickel concentration was above 9 ppm . The corrosion products also altered cell morphology, induced cell necrosis, and decreased cell numbers . The cell replication was inhibited at the G0-G1 to S transition phase . This was the first study to demonstrate the cytotoxicity of corrosion products of current nitinol stent wire on smooth muscle cells, which might affect the postimplantation neointimal hyperplasia and the patency rate of cardiovascular stents .

Akush Ginekol (Sofiia), 2000, 39(2), 3 - 6
{The basic problems and outlook for assisted reproductive technologies}; Kozovski I et al.; In this article the authors discuss some problems in ART:COH, stimulation protocols, culture media, transfer of embryos, luteal phase support, ethical, financial, religious and legal problems etc . Tha main complications are discussed, toosyndrom and pseudosyndrom of the empty follicle, OHSS, multiple pregnancy . In these cases the prophylactic and therapeutical measures are pointed out . Finally the main perspectives and developments of ART in the new millennium are outlined.

Thromb Res, 2000 Jul 15, 99(2), 173 - 8
Long-term effect of 17beta-estradiol and thrombin on tissue factor pathway inhibitor release from HUVEC; Bilsel AS et al.; In spite of the increasing evidence that estrogens have protective effects on the vascular system, the evidence that estrogens may contribute to the risk of thrombosis is still being debated . We investigated the effect of 17beta-estradiol (E2) on tissue factor pathway inhibitor (TFPI) release from of cultured human umbilical vein endothelial cells (HUVEC) . In this study HUVEC were harvested by collegenase treatment and cultured in multiwelled plates with medium 199 supplemented with 10% fetal calf serum and antibiotics . The cells were incubated in the presence or absence of E2 (1 and 100 nM) with/without thrombin (4 U/mL) for 6 or 24 hours . After the incubations TFPI level of media were measured by IMUBIND Total Eliza kit.Our results demonstrates that E2 at physiological concentrations decreases the release of TFPI from HUVEC significantly . Thrombin also decreases TFPI antigen levels detected in culture media . When combined with thrombin the effect of estrogen is not visible due the much higher effectivity of thrombin in diminishing TFPI levels . These results show that E2 shifts the hemostatic balance towards the procoagulant phase through lowering the TFPI levels secreted by the endothelium.

J Pharmacol Exp Ther, 2000 Sep, 294(3), 822 - 9
The effect of berberine chloride on experimental colitis in rats in vivo and in vitro; Zhou H et al.; Berberine is an isoquinoline alkaloid with multiple pharmacological actions, including anti-inflammatory activity . The aims of this study were to examine the effect of berberine on the mucosal healing process and to investigate whether berberine can inhibit the increased production of interleukin-8 in trinitrobenzene sulfonic acid-induced colitis in rats . Berberine was administered orally for 3 days or 1 week at a dosage of 7.5 or 15 mg/kg/day . Tissue damage scores, body weight, colon wet weight, and colon wall thickness were measured, and myeloperoxidase activity in colon tissue was also examined . Histological lesions, morphological damage, and myeloperoxidase activity were reduced after 1 week of treatment with berberine at a dosage of 15 mg/kg/day . Furthermore, 1 week after trinitrobenzene sulfonic acid treatment, the production of interleukin-8 by cultured rectal mucosa or cardiac blood mononuclear cells with or without stimulation of lipopolysaccharide for 24 h was also analyzed by enzyme-linked immunosorbent assay . Cardiac blood mononuclear cells and rectal mucosa of normal rats produced substantial amounts of interleukin-8, which increased strikingly with the stimulation of lipopolysaccharide . Cardiac blood mononuclear cells and rectal mucosa of trinitrobenzene sulfonic acid-treated rats secreted more interleukin-8 than those of normal rats . The addition of berberine with a concentration of 10(-5) M to the culture media resulted in an inhibition of interleukin-8 production of rectal mucosa.

J Vet Med Sci, 2000 Jul, 62(7), 725 - 9
Association of tightly spiraled bacterial infection and gastritis in pigs; Park JH et al.; Tightly spiral bacteria were observed only in the pyloric mucosa of 4 (8.0%) of 50 swine stomachs, mainly in the surface of epithelia, the gastric pits and the lumen of gastric glands . The presence of the spiral bacteria was significantly associated with chronic pyloric gastritis (p<0.05) . Mean gastritis score of the bacteria-positive pyloric mucosa was 3.25 +/- 0.25, whereas that of the bacteria-negative pyloric mucosa was 2.37 +/- 0.12 . Parakeratosis and hyperkeratosis were spontaneously seen in the mucosa layer of pars oesophagea, regardless of the bacterial infection . Marked infiltration of mononuclear cells and granulocytes were seen in the cardiac mucosa, regardless of the bacterial infection . Mean gastritis score of the bacteria-positive cardiac mucosa was 3.27 +/- 0.32, whereas that of the bacteria-negative cardiac mucosa was 2.84 +/- 0.13 . There was no significant difference between the bacteria-positive and negative cardiac mucosa (p>0.05) . Inflammatory response in the fundic mucosa was rare (gastritis score=0.75 +/- 0.08) . The tightly spiraled bactera were not cultured with various culture media . These results suggest that the presence of tightly spiraled bacteria is associated with only the pyloric gastritis in pigs.

J Cardiovasc Pharmacol, 2000 Aug, 36(2), 152 - 61
Interaction between monocytes and vascular smooth muscle cells enhances matrix metalloproteinase-1 production; Zhu Y et al.; Matrix metalloproteinase-1 (MMP-1) plays an important role in atherosclerotic plaque rupture . The purpose of this study was to investigate the expression of MMP-1 by cell-to-cell interactions between monocytes and vascular smooth muscle cells (VSMCs) . Human VSMCs and THP-1 cells (human monocytoid cells) were cocultured . MMP-1 levels were measured by enzyme-linked immunosorbent assay . Collagenolytic activity was determined by fluorescent labeled-collagen digestion . Immunohistochemistry was performed to determine which types of cells produce MMP-1 . Adding THP-1 cells to VSMCs markedly increased the MMP-1 levels and activity of the culture media . MMP-1 levels were maximal when the cellular ratio of THP-1 cells/VSMCs was 1.0 . Immunohistochemistry revealed that both types of cells in the coculture produced MMP-1 . Separated coculture experiments showed that both direct contact and a soluble factor(s) contributed to MMP-1 production . Neutralizing anti-interleukin (IL)-6 and tumor necrosis factor-alpha antibodies inhibited coculture conditioned medium-induced MMP-1 production by VSMCs and THP-1 cells . Protein kinase C inhibitors, tyrosine kinase inhibitors, and a mitogen-activated protein kinase inhibitor significantly inhibited MMP-1 production by cocultures . Direct cell-to-cell interaction between THP-1 cells and VSMCs enhanced MMP-1 synthesis in both types of cells . Increased local MMP-1 production and activity induced by monocyte-VSMC interaction play an important pathogenic role in atherosclerotic plaque rupture.

Vox Sang, 2000, 78 Suppl 2, 149 - 53
Expression of red cell surface antigens during erythropoiesis; Daniels G et al.; BACKGROUND AND OBJECTIVES: We have analysed the appearance and disappearance of cell surface markers during erythropoiesis, in vitro . MATERIALS AND METHODS: CD34+ haemopoietic progenitor cells were isolated from umbilical cord blood and cultured by three different methods, all in the presence of erythropoietin . The methods included two one-stage techniques, one serum-free, the other with serum present, and one serum-present two-stage method . RESULTS: The appearance of cell surface markers on the differentiating erythroid cells varied slightly from sample to sample, but differed more substantially between techniques, with the cells differentiating more rapidly in the culture media containing serum . The order of appearance of the markers, which was constant in the three methods, was as follows: glycophorin C, Kell, Rh-associated glycoprotein, glycophorin A, band 3, Rh proteins, and glycophorin B . CONCLUSION: Cell surface antigens can be used as markers for mapping the progress of erythroid differentiation during erythropoiesis.

Iowa Orthop J, 2000, 20, 11 - 6
Transforming growth factor beta one (TGF-beta 1) enhancement of the chondrocytic phenotype in aged perichondrial cells: an in vitro study; Lee MC et al.; BACKGROUND: Perichondrium is recognized as a tissue with chondrogenic potential yielding cells which can be used for osteochondral repair . Factors which influence the proliferative ability and chondrocytic phenotype of such cells include age and presence of specific growth factors, i.e . TGF-beta 1 . The present in vitro study assessed proliferation and markers of chondrocytic phenotype in cells extracted from the rib perichondrium of four- to five-year-old aged rabbits, and assessed the effects of exogenously added TGF-beta 1 on those cells . METHODS: Assays included 3H-thymidine incorporation (cell proliferation), 35S-sulfate incorporation (proteoglycan synthesis) and quantitative RT-PCR for determination of type II collagen gene expression . RESULTS: The results demonstrated that addition of TGF-beta 1 to the culture media stimulated thymidine incorporation and proteoglycan synthesis up to four- and five-fold, respectively, in aged perichondrium-derived cells . Moreover, the exogenous addition of TGF-beta 1 to the culture media resulted in an upregulation of transcriptional expression of the type II collagen gene . CONCLUSIONS: In summary, the present study has demonstrated that exogenously added TGF-beta 1 can stimulate proliferation and chondrocytic phenotype in aged perichondrium-derived cells in vitro.

Biotechnol Prog, 2000 Jul-Aug, 16(4), 657 - 60
High cell density culture of Yarrowia lipolytica using a one-step feeding process; Kim JW et al.; Yarrowia lipolytica is a potentially useful host for heterologous protein production . To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y . lipolytica, the effects of medium components and their concentrations on the growth of Y . lipolytica have been investigated . Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y . lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol . Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y . lipolytica up to 83 g/L dry cell weight in a batch culture . Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y . lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol) . The easy cultivation of recombinant Y . lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.

J Mol Med, 2000, 78(4), 217 - 27
Endogenous apolipoprotein E modulates cholesterol efflux and cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 and lipid-free apolipoproteins in mouse peritoneal macrophages; Langer C et al.; We investigated the effect of endogenous apolipoprotein (apo) E synthesis in mouse peritoneal macrophages on cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 (HDL3) and lipid-free apolipoproteins (apo) . After loading with acetylated LDL (acLDL) peritoneal macrophages from wild-type (apoE(+/+)) and apoE-deficient (apoE(-/-)) mice were incubated with medium alone or with liposomes, HDL3, lipid-free apoA-I, or lipid-free apoE3 . Cholesterol and cholesteryl esters in the cells and culture media were quantified by HPLC . Incubation of apoE(+/+) or apoE(-/-) macrophages for 18 h with medium alone or with liposomes did not cause significant changes in cellular cholesterol . Addition of HDL3, apoA-I, or apoE3 to the medium led to significant cholesterol efflux, which was less efficient in apoE(-/-) macrophages than in apoE(+/+) macrophages . HDL and lipid-free apolipoproteins were more effective in reducing the cellular content of cholesteryl esters of apoE(+/+) macrophages than of apoE(-/-) macrophages, suggesting that endogenous apoE stimulates cholesteryl ester hydrolysis . The difference in the mass of cholesteryl esters was more pronounced for cholesteryl arachidonate and linoleate than for cholesteryl oleate or palmitate . Furthermore, in {(14)C}arachidonate labeling experiments cholesterol arachidonate hydrolysis was higher in apoE(+/+) macrophages than in apoE(-/-) macrophages in the presence of cholesterol efflux mediated by HDL3 or apoA-I . In contrast, in the absence of cholesterol efflux cholesterol arachidonate synthesis was higher in apoE(+/+) macrophages than in apoE-/- macrophages . Taken together, our data suggest that endogenous apoE stimulates cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by HDL3 and lipid-free apolipoproteins in mouse peritoneal macrophages . This may contribute to the antiatherogenic effect of apoE.

J Endocrinol, 2000 Aug, 166(2), 437 - 45
Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells: a specific assessment by capillary electrophoresis; Arcelloni C et al.; The aim of this study was to compare the metabolic pathway to mature insulin through the intermediate forms (32-33 split, 65-66 split, des31,32 and des64,65) in human or murine cells engineered for the release of wild-type human proinsulin and in a genetically mutated one, in the search for a new approach for an insulin-dependent diabetes mellitus cure by gene therapy . Primary human fibroblasts, myoblasts and stabilized cell lines (HepG2 and NIH3T3) were transduced either with a retroviral vector coding for wild-type proinsulin or for a genetically mutated one, carrying cleavage sites sensitive to furin . The pattern of all the proinsulin cleavage products released into the cell culture supernatants was analyzed by capillary electrophoresis . All the cells transduced with the wild-type gene released intact proinsulin . HepG2 released a considerable amount of 65-66 split and des64,65, while primary myoblasts released all the intermediate forms and a limited amount of mature insulin . All the cells transduced with a furin-sensitive proinsulin gene released a higher amount of mature insulin (23-59% conversion yield) than the cells expressing wild-type proinsulin, whereas the total insulin was nearly constant . Only primary cells released all the cleavage products . Screening a wide variety of non-endocrine cells has revealed a large difference in the processing and release of immature and mature insulin forms, pointing to human hepatic cells as the most efficacious . Capillary electrophoresis provided on-line and in a single run a complete overview of the proinsulin metabolic pathway in different cells.

Zh Mikrobiol Epidemiol Immunobiol, 2000 May-Jun, (3), 29 - 31
{The development and testing of dried nutrient media for the demonstration of ureaplasmas}; Akhmedova EM et al.; The results of the development of new culture media for the indication of ureaplasmas are presented . The media have been developed on the basis of the dried nutrient broth produced by the Research and Production Amalgamation "Nutrient Media" . The composition of the media includes Russian-made ingredients ensuring the growth of ureaplasmas . The physico-chemical and biological characteristics of the media have been perfected . The clinical trial of the newly developed media has been carried out in the process of the examination of 280 pregnant women with the normal course of pregnancy, with the threat of pregnancy interruption and with complications of pregnancy.

Endocrinology, 2000 Aug, 141(8), 2877 - 85
Antigen-presenting cells in the female reproductive tract: influence of estradiol on antigen presentation by vaginal cells; Wira CR et al.; The objective of the present study was to define the afferent arm of the mucosal immune system in the lower female reproductive tract . We report here that antigen presentation by vaginal cells is under hormonal control . When vaginal cells from ovariectomized rats treated with estradiol (0.01-10 microg) were incubated with ovalbumin-specific T cells and ovalbumin, a dose-dependent inhibition of antigen presentation was measured . In time course studies, estradiol given to ovariectomized rats inhibited vaginal cell antigen presentation within 24 h after a single injection, relative to that seen in saline controls . To determine whether changes in antigen presentation were attributable to the effect of estradiol on the number of antigen-presenting cells (APCs) in the vagina, tissues were analyzed by immunohistochemistry . Our findings indicate that estradiol inhibited antigen presentation without affecting the number of major histocompatibility complex class II positive cells and at a time when macrophage/dendritic cells/granulocytes in the vagina increase in response to estradiol treatment . Antibody neutralization studies indicated that antigen presentation by vaginal cells from ovariectomized rats is mediated through class II and involves the expression of transmembrane proteins B7.1 and B7.2 . In other studies, vaginal APCs interact with thymus APCs to synergistically enhance antigen presentation under conditions in which vaginal antigen presentation is inhibited by estradiol . Analysis of conditioned media indicates that enhancement of thymus antigen presentation involves the release of a soluble factor(s) into the culture media of vaginal cells . When spleen cells were cocultured with vaginal cells from saline-treated rats, proliferation increased in the presence of concanavalin A and/or phytohemagglutinin and decreased with lipopolysaccharide, relative to spleen cells and mitogen alone . In contrast, when incubated with vaginal cells from estradiol-treated rats, spleen cell proliferation was not affected with concanavalin but was inhibited with phytohemagglutinin and lipopolysaccharide . These studies demonstrate that estradiol regulates antigen presentation by vaginal cells and that vaginal cells, in turn, influence antigen presentation, as well as B and T cell proliferation.

Vet Surg, 2000 Jul-Aug, 29(4), 347 - 57
In vitro evaluation of the effect of dimethyl sulfoxide on equine articular cartilage matrix metabolism; Smith CL et al.; OBJECTIVE: To evaluate the effects of dimethyl sulfoxide (DMSO) on equine articular cartilage matrix metabolism . STUDY DESIGN: Using a cartilage explant culture system, proteoglycan (PG) synthesis, PG release, lactate metabolism, chondrocyte viability, and metabolism recovery were determined after cartilage exposure to DMSO . SAMPLE POPULATION: Cartilage harvested from metacarpophalangeal and metatarsophalangeal joints of 12 horses (age range, 1 to 10 years) . METHODS: Explants were exposed to concentrations of DMSO (1% to 20%) for variable times (3 to 72 hours) . PG synthesis and release were determined by a radiolabel incorporation assay and dimethylmethylene blue (DMMB) dye assay, respectively . Lactate released into culture media was measured, and chondrocyte viability was assessed using the Formizan Conversion Assay and a paravital staining protocol . Metabolism recovery was assessed in explants that were allowed to recover in maintenance media after exposure to DMSO . RESULTS: PG synthesis and lactate metabolism were inhibited in a dose- and time-dependent manner after exposure to DMSO concentrations > or = 5%; there was no significant alteration in PG release . No change in chondrocyte viability was detected after incubation with DMSO . PG synthesis and lactate metabolism returned to baseline rates when allowed a recovery period after exposure to DMSO . CONCLUSIONS: DMSO concentrations > or = 5% suppress equine articular cartilage matrix metabolism . Suppression of PG synthesis and lactate metabolism is reversible and does not appear to be the result of chondrocyte death . CLINICAL RELEVANCE: Equine clinicians adding DMSO to intraarticular lavage solutions should be aware that DMSO may have deleterious effects on equine articular cartilage matrix metabolism.

Virchows Arch, 2000 Jun, 436(6), 560 - 6
Evidence of M cells as portals of entry for antigens in the nasopharyngeal lymphoid tissue of humans; Fujimura Y; The nasopharyngeal tonsils (adenoids) are prominent components of human nasal-associated lymphoid tissues (NALT) . However, the role of the nasopharyngeal tonsils in antigen uptake for initiation of the mucosal immune response is unknown . The aims of this study were to describe the ultrastructure and function of the M cells of the human nasopharyngeal tonsils and to clarify their capacity for antigen uptake . Tissues obtained from eight patients undergoing adenectomy were examined by light and electron microscopy . Lymphoepithelium covers the nasopharyngeal lymphoid tissue and consists of ciliary epithelium, non-ciliary epithelial cells, M cells, goblet cells, and many intraepithelial lymphoid cells . M cells have irregular and broad cytoplasm-containing microvilli on their surface and small vesicles in their cytoplasm . Many lymphoid cells were enfolded by M cells . The uptake of horseradish peroxidase (HRP) in the tissue in organ culture was studied using histochemical techniques . Excised adenoid tissue was incubated in RPMI 1640 culture media with HRP for 10, 30, and 60 min . HRP which had adhered to the surface was taken up in vesicles and then transported in vesicles and tubules by M cells . The M cells of nasopharyngeal lymphoid tissue were ultrastructurally and functionally similar to those in human Peyer's patches and colonic lymphoid follicles . These findings indicate that NALT bears similarities to the gut-associated lymphoid tissue, and its antigen uptake capacity may be important for initiation of immunity in the upper aerodigestive tract.

Zhonghua Zhong Liu Za Zhi, 1998 Jul, 20(4), 267 - 9
{A study on the correlation between the activity of 72,000 type IV collagenase and the metastatic potential of cancer cells}; Gao Q et al.; OBJECTIVE: To evaluate the enzyme activity of 72,000 type IV collagenase and its relationship with the metastatic potential of cancer cells . METHODS: The levels of secreted 72,000 type IV collagenase in the conditioned media of five human cancer cell lines with different metastatic potential and a normal lung fibroblast strain treated with cancer cell culture media were examined by gelatin zymography and densitometric analysis . RESULTS: The levels of 72,000 type IV collagenase secreted by cancer cells with high metastatic potential (PG, WM451 and WM983a) were higher than those secreted by cancer cells with low metastatic potential (PAa and WM35) . In the conditioned media of fibroblasts which were treated with the culture media of PG and WM451, enhanced levels of activation were observed . CONCLUSION: The secretion of 72,000 type IV collagenase is closely correlated to the metastatic potential of cancer cells . The cancer cells with high metastatic potential may possibly through certain soluble mediators stimulate normal fibroblasts to activate 72,000 type IV procollagenase.

Endocr Regul, 1997 Sep, 31(3), 157 - 161
Effect of luteinization stimulator and androgens on maturation of porcine granulosa cells; Vranova J et al.; Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, enhanced both basal and gonadotropins-stimulated production of progesterone (P4) by immature granulosa cells . The activity of LS was found in cell conditioned media (CM) obtained after the 3-day cultivation of preovulatory granulosa cells . Influence of testosterone, androstenedione and dihydrotestosterone on LS-enhanced P4 secretion was tested in culture of granulosa cells isolated from small follicles (1-3 mm) . Small porcine granulosa cells were cultivated with or without LS in the presence of testosterone, androstenedione and dihydrotestosterone in concentration 10-10, 10-8 and 10-6 mol.l-1 . In the absence of LS, P4 production in the media with androgens was not significantly different from controls . LS alone significantly enhanced progesterone production by SGC . Androgens present in the culture media together with LS decreased a stimulatory influence of LS on P4 secretion . These data suggest a possible modulation of granulosa cells maturation by androgens.

Kidney Int, 2000 Aug, 58(2), 881 - 8
Bradykinin and nitric oxide generation by dialysis membranes can be blunted by alkaline rinsing solutions; Coppo R et al.; BACKGROUND: Bradykinin (BK) generation following the first contact of blood with the dialysis materials is thought to enhance hypersensitivity reactions (HSRs) . Some of the effects of BK are mediated by nitric oxide (NO) . We have recently reported that the pH of diluted blood modulates the kinin system . The present study was aimed to investigate the role of the pH of culture media and filter-washing solutions and BK and NO generation, either in vitro and ex vivo . METHODS: BK was measured by a specific enzyme-linked immunosorbent assay (ELISA), and NO synthase (NOS) activity by 3H-citrulline production after incubation with 3H-arginine and nitrites by using the Griess reagent . In in vitro experiments, NOS activity was detected in endothelial cells (ECs) cultured with graded BK concentrations at various pH values . Blood from 30 patients in regular dialysis was ex vivo circulated in one single passage through minifilters prerinsed with pH 7 or pH 8 phosphate buffer (PB) solutions . The out-flowing blood was tested for BK and nitrite content and was incubated with cultured ECs to evaluate its capacity to modulate NOS activity . RESULTS: BK induced in vitro a dose-dependent increase in NOS activity of ECs, which was mediated by tyrosine kinase phosphorylation . NO generation was enhanced at pH 7.2, which remained unchanged at pH 7.6 . In ex vivo experiments, blood out-flowing after one passage on filters washed with pH 7 PB solutions had increased BK levels (P < 0.0001), increased nitrites (P < 0.05), and enhanced EC NOS activity (P < 0 . 05) in comparison to data found when filters were washed with pH 8 PB . Only when the filters were rinsed with a solution at pH 7 did PAN DX and AN69 membranes show a distinct BK generation capability, and cuprophane a peculiar capability to enhance NOS . Such effects were prevented when dialyzers were prerinsed with pH 8 PB . Multiple regression analysis showed that the pH of the uremic blood was the driving factor for BK and NOS activation (r = 0.54, P < 0.02) . CONCLUSIONS: BK and NO generation are modulated by environmental pH . Rinsing the blood and dialysate compartments of filters with an alkaline solution prior to use may mitigate the activation of mediators likely to be involved in some HSRs.

Biochem, Eng . J. . 2000 Aug 1, 6(1), 13 - 18
Biomass production and biochemical variability of the marine microalga Isochrysis galbana in relation to culture medium; Sanchez S et al.; We have studied the autotrophic growth of the marine microalga, Isochrysis galbana Parke, in a batch photobioreactor, comparing five different culture media and analysing the influence of each on growth kinetics as well as on the fatty-acid composition and protein content of the biomass . All the experiments were performed at 15 degrees C, with the culture medium at pH 8.0, a specific rate of air supply of 1vv(-1)min(-1) and a continuous illumination of 40-43Wm(-2) . The results show no parallel between good nutritional characteristics and high values of the kinetic parameters . Nevertheless, a compromise between the nutritional factors and growth kinetics could be provided by Ukeles medium, which provided a biomass with a good composition in polyunsaturated fatty acids (quotient n3/n6=3.2), an adequate protein content (25.3%) and relatively high values, although not the highest registered, for maximum specific growth rate (micro(m)=0.018h(-1)) and biomass productivity (1.9x10(-3)kgm(-3)h(-1)).

Plant Sci, 2000 Jul 14, 156(1), 85 - 94
High frequency of cytogenetic aberration in transgenic oat (Avena sativa L.) plants; Choi H et al.; Cytological abnormalities were observed in transgenic oat (Avena sativa L . cv . GAF/Park-1) produced by microprojectile bombardment of mature seed-derived highly regenerative tissues . Of the plants from 48 independent transgenic lines examined, plants from only 20 lines (42%) were karyotypically normal (2n=6x=42) without detectable chromosomal aberrations; plants from 28 lines (58%) had chromosomal variation, i.e . aneuploids and structural changes . No significant difference in cytological aberration was observed between the two different culturing systems used for transformation: 57% chromosomal abnormalities in plants derived from D'BC2 medium (2.0 mg/l 2,4-D, 0 . 1 mg/l BAP and 5.0 microM cupric sulfate) used for tissue initiation and maintenance and 60% in plants from tissue initiated on D'BC2 and maintained on DBC3 (1.0 mg/l 2,4-D, 0.5 mg/l BAP and 5.0 microM cupric sulfate) . Comparative differences in chromosomal status frequently occurred among plants regenerated from the same T(0) line . The most common cytological aberration in transgenic plants was aneuploidy, followed by deletion of chromosomal segments; no change in ploidy level was observed . In contrast, nontransgenic plants, regenerated from tissues comparable in age and culture media to that used for transgenic tissues, had a much lower percentage of karyotypic abnormality (0-14%) . Our data indicate that some stress(es) imposed by the transformation process, e.g . osmotic treatment, bombardment and selection, leads to cytological variation in transgenic oat plants, an observation similar to that observed in our recent studies with transgenic barley plants.

Thyroid, 2000 Jun, 10(6), 481 - 7
Thyroid organoid formation in simulated microgravity: influence of keratinocyte growth factor; Martin A et al.; The generation of artificial human thyroid tissues in suspension (low-shear environment, present in simulated microgravity {MG} and generated by a rotary cell culture system {RCCS}), was enhanced by increasing medium kinematic viscosity with a (3% v/v) suspension of extracellular matrix (basement membrane extract {BME}) in serum-free medium to generate artificial human thyroid organoids . Recombinant human keratinocyte growth factor (KGF, 7 ng/mL) facilitated human thyrocyte aggregation and three-dimensional (3-D) differentiation . There was an MG-associated decrease in extractable DNA that was reversed after addition of keratinocyte growth factor (KGF) . In simulated MG, the increase in extractable DNA after KGF addition was up to 170% over non-KGF control cultures . In contrast, monolayer cultures in unit gravity showed a maximum DNA increase of 39% after KGF addition . Morphologically, differentiated thyroid neofollicles displayed polarization and were located in close proximity after 2 weeks of culture . Immunogold labeling with antibody to human thyroglobulin (Tg) revealed staining of follicular lumina and secretory vesicles, and a time-dependent increase in human Tg was detected in the culture media . Culture under simulated MG thus allowed direct visualization of KGF-facilitated thyrocyte/extracellular matrix interaction . Such artificial human thyroid organoids-generated in MG and in the presence of KGF-structurally resembled natural thyroid tissue . The above findings may have implications for autoimmune thyroid disease where KGF (if, for example, secreted locally by intraepithelial gammadelta T cells among other cells) may contribute to thyroid cell growth.

Toxicol In Vitro, 2000 Aug, 14(4), 297 - 307
Protection by free oxygen radical scavenging enzymes against salicylate-induced embryonic malformations in vitro; Karabulut AK et al.; Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals . In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated . Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr . ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml . Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 micromol/ml) . The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations . When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner . There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud . When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations . Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL . There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione . The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid . An enhanced production of these metabolites in embryonic tissues may be directly related to the increased risk of congenital malformations.

Toxicol Lett, 2000 Jul 27, 116(1-2), 45 - 9
Effect of lead on Sertoli-germ cell coculture of rat; Adhikari N et al.; Mixed cultures of Sertoli and germ cells were prepared from rat testes and their response to lead (Pb) was studied . Cultures consisted of a monolayer of Sertoli cells to which clusters of germ cells were attached . The effect of Pb added as lead acetate was tested at 0.0, 0.4, 4.0 and 40.0 microM for 24 and 48 h intervals . Addition of Pb to the culture medium caused germ cells to progressively detach from the Sertoli cell monolayer into the medium in a concentration and duration dependent manner Viability of the detached cells as judged by trypan blue exclusion test showed a decrease with increase in time and concentration of Pb . Significant leakage of lactate dehydrogenase (LDH) was recorded in the culture media only at the higher concentrations of 4.0 and 40.0 microM . Thus Pb at the doses tested induced cytotoxicity in rat Sertoli-germ cell coculture.

Hong Kong Med J, 2000 Jun, 6(2), 163 - 8
Laboratory aspects of assisted reproduction; Yeung WS et al.; A number of advances have been made concerning the laboratory aspects of assisted reproduction . Intracytoplasmic sperm injection has revolutionised the treatment of male infertility . With the development of better embryo culture media, blastocyst transfer is now possible and is likely to reduce high-order multiple pregnancy in assisted reproduction treatment . Pre-implantation genetic diagnosis has become an alternative to prenatal diagnosis . The recent use of molecular biology techniques to detect small genetic defects in men with severe male-factor infertility has provided information for the better counselling of these patients . Other techniques that are being developed are likely to have a tremendous impact on assisted reproduction treatment . These include in vitro maturation, follicle culture, and oocyte/ovarian tissue cryopreservation . The current status of the developments in the laboratory aspects of assisted reproduction is reviewed in this article.

Invest Ophthalmol Vis Sci, 2000 Jul, 41(8), 2170 - 6
The effect of hypoxia on endogenous corneal epithelial eicosanoids; Mieyal PA et al.; PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways . The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate . METHODS: Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia . After culture, PGE2 in media was quantitated by enzyme immunoassay . 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry . COX-1 and -2 protein expression in corneal epithelium was determined by Western blot . Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of {14C}AA to {14C}PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection . RESULTS: In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher . Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis . Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture . Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE . CONCLUSIONS: Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced . Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.

Neurosci Lett, 2000 Jul 21, 288(3), 203 - 6
Effects of sodium azide on the secretion of soluble amyloid-beta precursor protein and the accumulation of beta-amyloid(1-40) in cultured chick neurons; Hedin HL et al.; Sodium azide has been reported in the literature to reduce the release of secreted amyloid beta-precursor protein (AbetaPPs) and to produce a large increase in the cellular level of an 11.5 kDa C-terminal AbetaPP derivative containing the beta-amyloid (Abeta) sequence . Here we report that 1 mM of sodium azide, reduced the constitutive AbetaPPs secretion from cultured embryonic chick neurons after 12 h of incubation . After 24 h of incubation there was a modest increase in lactate dehydrogenase (LDH) release and no change in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, suggesting that the reduced AbetaPPs secretion was not due to the cell toxic effects of NaN(3) . However, NaN(3) reduced the accumulation of Abeta(1-40) in the cell lysates and decreased the acetylcholine esterase activity both in cell culture media and in cell lysates . It is concluded that the effect of NaN(3) upon AbetaPP metabolism in the chick cultured neurons may be a rather non-specific effect.

Jpn J Pharmacol, 2000 May, 83(1), 56 - 62
Effects of angiotensin-converting enzyme inhibitors on spontaneous or stimulated generation of reactive oxygen species by bronchoalveolar lavage cells harvested from patients with or without chronic obstructive pulmonary disease; Teramoto S et al.; We examined the effects of angiotensin-converting enzyme (ACE) inhibitors on spontaneous or stimulated generation of reactive oxygen species (ROS) by bronchoalveolar lavage (BAL) cells prepared from 6 patients with chronic obstructive pulmonary disease (COPD) and from age-matched control subjects without COPD . The ROS produced by BAL cells were measured by the lucigenin-dependent chemiluminescence method . The application of ACE inhibitors into culture media containing BAL cells inhibited spontaneous and stimulated generation of ROS by BAL cells from COPD patients and control subjects in an ambroxol-concentration-dependent manner . Alacepril, an ACE inhibitor bearing SH-group, inhibited the oxygen radical production and generation by BAL cells from COPD patients in a dose-dependent fashion . Approximately 0.6-0.7 mM of alacepril inhibited 50% of the ROS production by BAL cells from COPD patients, whereas a slightly higher concentration (3 mM) of lisinopril, an ACE inhibitor not bearing an SH-group, was necessary to inhibit the production of ROS . These results suggest that an ACE inhibitor may act as an pulmonary antioxidant in patients with COPD.

Kidney Int, 2000 Jul, 58(1), 43 - 50
Hypoxia and interleukin-1beta stimulate vascular endothelial growth factor production in human proximal tubular cells; El Awad B et al.; BACKGROUND: Vascular endothelial growth factor (VEGF) promotes angiogenesis and inflammatory reactions . VEGF mRNA is detectable in the proximal tubules of inflamed kidneys but not in normals . In other organs VEGF gene expression is induced by hypoxia and cytokines such as interleukin 1 (IL-1) . To identify the cellular mechanisms in control of tubular VEGF production, we studied effects of hypoxia and IL-1beta in VEGF mRNA levels, VEGF secretion, and activity of the hypoxia-inducible dimeric transcription factor 1 (HIF-1alpha/beta) in human proximal tubular epithelial cells (PTECs) in primary culture . METHODS: PTECs were grown in monolayers from human kidneys . Hypoxia was induced by incubation at 3% O2 . VEGF mRNA was quantitated by competitive polymerase chain reaction following reverse transcription . VEGF was measured by enzyme-linked immunoassay . HIF-1alpha was demonstrated by Western blot analysis and HIF-1 DNA binding by gel shift assay . RESULTS: Significant amounts of VEGF mRNA and VEGF protein were measured in PTEC extracts and culture media, respectively . Stimulation of VEGF synthesis at low O2 tension and following IL-1beta treatment was detectable at the protein level only . Nuclear HIF-1alpha protein levels and HIF-1 binding to DNA were also increased under these conditions . CONCLUSIONS: PTECs in culture produce VEGF . One mechanism of induction appears to be increased DNA binding of HIF-1 to hypoxia-responsive elements in the VEGF gene promoter . In inflammatory diseases of the kidney, tubular cell-derived VEGF may contribute to microvascular leakage and monocyte extravasation.

J Invest Dermatol, 2000 Jul, 115(1), 118 - 23
Expression patterns of placenta growth factor in human melanocytic cell lines; Graeven U et al.; Expression patterns of the angiogenic placenta growth factor and its receptor neuropilin-1 were assessed in normal human melanocytes, SV40T-transformed melanocytes, and melanoma cells derived from primary and metastatic lesions . As determined by reverse transcription-polymerase chain reaction all primary and metastatic melanoma cell lines tested and SV40T-transformed melanocytes coexpressed two placenta growth factor splice variants (placenta growth factor-1 and -2) as well as neuropilin-1 mRNA . Placenta growth factor protein was detected in conditioned media derived from five of eight melanomas and from SV40T-transformed melanocytes . In contrast to melanoma cells, normal melanocytes did not express placenta growth factor mRNA at detectable levels and did not secrete placenta growth factor protein . By contrast, neuropilin-1 transcripts were detected in some of the normal melanocytes . Secretion of placenta growth factor by melanoma cells appeared to be constitutive because it was not affected by the addition of exogenous growth factors including insulin, epidermal growth factor, or basic fibroblast growth factor to culture media . Although melanoma cells expressed both, neuropilin-1 and flt-1, exogenous homodimeric placenta growth factor had no effect on melanoma cell growth . Similarly, placenta growth factor did not induce urokinase-type plasminogen activator production in these cells . These findings demonstrate that melanoma progression is accompanied by deregulated, constitutive placenta growth factor expression . Placenta growth factor, however, serves no apparent autocrine role in melanoma proliferation . Further studies are needed to define the relative contribution of placenta growth factor to the angiogenic properties of human melanomas.

Rev Argent Microbiol, 2000 Apr-Jun, 32(2), 53 - 62
Factors influencing protease production by two Antarctic strains of Stenotrophomonas maltophilia; Vazquez SC et al.; The influence of culture medium buffer capacity, the supplementation of culture medium with L-ala and the requirement of calcium for exoprotease production by Antarctic psychrotrophic Stenotrophomonas maltophilia strains ANT-1-1 and ANT-7-1 were examined . When increasing concentrations of calcium chloride (0 to 0.3 g l-1) were added to culture media, maximum protease production yields increased 70-75% (ANT-1-1) and 50% (ANT-7-1), while biomass levels showed little difference . Calcium was also necessary for optimal activity of proteases . L-ala had no effect on protease production . The reduction in buffer capacity, with the consequent change in external pH, had a positive effect, enhancing protease yields . Secretion of proteases into the medium started at the beginning of the stationary phase, corresponding with a rise in pH values up to pH 8.7 and was maximal at 36 h of culture . These results indicate that the regulation of calcium concentration and buffer capacity and also pH monitoring are factors to be considered when the design of an industrial culture medium and the optimisation of protease production processes using these Antarctic strains are concerned.

Histochem Cell Biol, 2000 May, 113(5), 349 - 61
Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors; Klingler C et al.; Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway . Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity . In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004 . Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers . The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected . In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved . Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.

Rev Biol Trop, 1999 Sep, 47(3), 381 - 91
{Cyanobacteria as indicators of organic contamination}; Peinador M; In two Costa Rican rivers used as receptors for domestic sewage, treated by primary stabilization ponds, were taken a total of 28 samplings located at the pond exit and at three different sites in each river: 100 m before the ponds discharge, at the discharge and 100 m after the discharge . These sampling were done for a five and a half years including dry and rainy seasons . In each sampling site, samples were collected of five different substrates: stones, submerge and semi submerge vegetation, tree trunks or sticks, water and artificial substrates . For each sample were used two types of artificial cultures, WC and BG110 . A total of 55 cyanobacteria species isolations were obtained, belonging to a 26 genera, between these the most common were Phormidium with nine species, Microcystis with five species, Leptolyngbya and Pseudanabaena with four species each and Oscillatoria with three species . More cyanobacteria species were isolated in water substrate and less isolations in tree trunks and submerge vegetation . Konvophoron, Cyanarcus and Pilgeria only were isolate from water samples inoculated in culture media WC and in few opportunities, while three Leptolyngbya species and four Phormidium species were isolated very often . At the stabilization ponds Phormidium sp4 was dominant in 25 of 28 sampling while in the last others were the chlorophycea I . In this study were observed an increase in the frequency of cyanobacteria at the higher contamination places, and a species substitution between different sampling points . There were no biomass studies, therefore is not possible to relate between different cyanobacteria species and some specific types of water quality.

Prostate, 2000 Jul 1, 44(2), 124 - 32
Autocrine effect of DHT on FGF signaling and cell proliferation in LNCaP cells: role of heparin/heparan-degrading enzymes; Kassen AE et al.; BACKGROUND: LNCaP cells are androgen-sensitive human prostate cancer cells . They are characterized by a bell-shaped growth curve in response to increasing doses of dihydrotestosterone (DHT) in culture . At a low concentration of DHT (0.1 nM), these cells show an increase in proliferation, but their growth is arrested at a high concentration (100 nM) of DHT . Results of our previous study demonstrated that the inhibitory effect of DHT at a high concentration was mediated through the action of TGF-beta1 . The objective of the present study was to elucidate the mechanism of the proliferative effect of DHT in LNCaP cells . METHODS AND RESULTS DHT stimulated LNCaP proliferation only when cells were cultured in the presence of serum . In serum-free cultures, the characteristic DHT-induced proliferation was not observed . The addition of neutralizing antibody against FGF-2 (basic fibroblast growth factor) was able to inhibit this DHT-induced proliferation . These results suggest that the proliferative effect of DHT was mediated through the action of FGF-2 . However, results of the reverse transcriptase polymerase chain reaction indicated that LNCaP cells did not express FGF-2 message . As a result, the source of FGF-2 in these cultures must be the serum supplemented in the culture media . FGF-2 can bind to heparin sulfate chains within the extracellular matrix (ECM) . In cultures treated with exogenous heparin, the proliferative effect of DHT was abolished . These results led to the development of the hypothesis that DHT treatment mediates the release of FGF-2 entrapped in the ECM through increased heparinase activity . The addition of heparinase to cultures of LNCaP cells, in the absence of DHT, was able to stimulate cell proliferation . Moreover, 0.1 nM DHT caused a significant increase in heparinase activity . CONCLUSIONS: These results provide a possible mechanism for DHT action in LNCaP cells . In the absence of DHT, FGF-2 in culture was trapped in the extracellular matrix and was not available to interact with LNCaP cells . However, in the presence of 0.1 nM DHT, heparinase activity in the culture was elevated and, as a result, it liberated the trapped FGF-2 which, in turn, stimulated proliferation in LNCaP cells .

Cell Mol Biol (Noisy-le-grand), 2000 Jun, 46(4), 785 - 95
Differential effects of zinc on amyloid precursor protein (APP) processing in copper-resistant variants of cultured Chinese hamster ovary cells; Borchardt T et al.; Previous studies have demonstrated that adding copper to Chinese-hamster ovary (CHO) cells greatly reduced the levels of beta-amyloid (Abeta) peptide in parental CHO-K1 and in copper resistant CHO-CUR3 cells which have lower intracellular copper levels . In the current study, zinc, the zinc chelator 1,10-phenanthroline or copper chelators bathocuproine and D-penicillamine were added to the culture media of stably transfected CHO cells . The data show that zinc up to concentrations of 50 microM or the presence of 1,10-phenanthroline specifically increased the level of secreted APP in CHO-K1 cells . By contrast, the level of secreted APP in CHO-CUR3 cells remained unaffected . APP holoprotein increased dramatically in CHO-CUR3 cells compared with CHO-K1 cells . The large decrease of Abeta release seen in both cell lines at elevated extracellular zinc levels was due to specific inhibition of secretion . These results indicate that a disturbed zinc-homeostasis may be an important factor influencing APP production, transport and processing.

Carcinogenesis, 2000 Jul, 21(7), 1403 - 9
The role of cyclooxygenase enzymes in the growth of human gall bladder cancer cells; Grossman EM et al.; Information suggests that the cyclooxygenase (COX) metabolites, the prostanoids, play a role in gall bladder physiology and disease . Non-steroidal anti-inflammatory drugs which inhibit COX enzymes have been shown in vivo and in vitro to alter the growth patterns of intestinal epithelial cells, and specific COX-2 inhibitors have been shown to decrease mitogenesis in intestinal epithelial cells . The present study was intended to evaluate the effect of specific COX inhibitors on the growth patterns of gall bladder cancer cells . Employing a human gall bladder cancer cell line, mitogenesis, apoptosis and prostaglandin E(2) (PGE(2)) formation were evaluated in response to serum and hepatocyte growth factor and transforming growth factor alpha stimulation in the presence and absence of specific COX-1 and -2 inhibitors . The effect of the mitogens on COX enzyme expression was also evaluated . Serum and the growth factors increased COX enzyme expression and mitogenesis, and decreased apoptosis as evaluated by the percentage of cells that were floating in culture media rather than attached . There was more DNA degradation in floating than in attached cells . The specific COX-2 inhibitor, but not the COX-1 inhibitor, decreased mitogenesis and increased gall bladder cell apoptosis as evaluated by the number of floating versus attached cells and the number of floating cells in the terminal phase of apoptosis or dead . The inhibition of mitogenesis and the increased apoptosis produced by the COX-2 inhibitor was associated with decreased PGE(2) production . The inhibition of replication of gall bladder cancer cells and the increase in apoptosis produced by the selective COX-2 inhibitor suggests that the COX enzymes and the prostanoids may play a role in the development of gall bladder cancer and that the COX-2 inhibitors may have a therapeutic role in the prevention of gall bladder neoplasms.

Eur J Endocrinol, 2000 Jul, 143(1), 133 - 8
Human GnRH-secreting cultured neurons express activin betaA subunit mRNA and secrete dimeric activin A; Florio P et al.; OBJECTIVE: To evaluate the expression of activin betaA-subunit mRNA and the secretion of activin A in/from cultured GnRH-secreting neuronal cells cloned from human olfactory epithelium (FNC-B4), which showed biochemical and antigenic properties of GnRH-secreting neurons . DESIGN: FNC-B4 cells were cultured in basal and conditioned media . METHODS: Reverse transcription-polymerase chain reaction (RTR-PCR) evaluated the expression of activin betaA-subunit mRNA . By using a specific ELISA, dimeric activin A concentrations were measured in culture media, in the absence or presence of carvone or forskolin and with different doses of progesterone, GnRH, and estradiol . RESULTS: RT-PCR experiments performed on total RNA isolated from FNC-B4 cells, using specific primers for the activin betaA gene, showed a 787bp DNA band corresponding to the betaA gene . FNC-B4 cells secreted activin A, and the highest accumulation in conditioned medium was achieved after 3h culture: the addition of forskolin, but not of carvone, was able to stimulate the release of activin A from cultured neuronal cells (P<0.01) . When progesterone or GnRH was added, a significant accumulation of activin A was observed (P<0.01), while estradiol administration did not significantly affect activin A secretion . CONCLUSION: To date, this is the only study, in an in vitro human model reporting, that GnRH-secreting neuronal cells expressed activin betaA-subunit mRNA, and released dimeric activin A in culture medium . The expression and secretion of activin suggests that in these cells activin A might exert its action by autocrine/paracrine mechanisms.

Int Arch Allergy Immunol, 2000 May, 122 Suppl 1, 44 - 9
Expression of eotaxin by normal airway epithelial cells after influenza virus A infection; Kawaguchi M et al.; BACKGROUND: Viral infection is known to cause lung inflammatory disease, including bronchial asthma . The mechanisms of inflammatory cell accumulation into the airways after viral infection are not well understood . Eotaxin is a CC chemokine which is a potent and specific agonist for CC chemokine receptor 3 (CCR3) . CCR3 is expressed on eosinophils, basophils and T lymphocytes . These cells are known to be key cells in the pathogenesis of asthma . Although it has recently been demonstrated that airway epithelial cells express eotaxin in vivo and in vitro, there are few data about its epxression in viral infection . We hypothesized that eotaxin may play an important role in attracting inflammatory cells to the airways after viral infection, and analyzed whether viral infection attracts eotaxin in bronchial epithelial cells in vitro . METHODS: Human airway epithelial cells obtained from bronchial tissue at lobectomy for lung cancer were infected with influenza virus A (subtype H3N2) . The cells and cultured media were collected 8, 24, and 48 h after infection . Eotaxin mRNA was analyzed with reverse transcriptase-polymerase chain reaction . Eotaxin protein levels in the culture media were analyzed by enzyme-linked immunosorbent assay . We also studied a blocking assay to analyze the intervention of proinflammatory cytokines in its production induced by influenza virus . RESULTS: Eotaxin mRNA appeared to be expressed constitutively in uninfected cells but was expressed more clearly in infected cells . Eotaxin protein release into culture media significantly increased after infection . Anti-TNF-alpha and anti-IL-1beta antibodies did not alter the eotaxin protein levels after viral infection . CONCLUSIONS: These results suggest that influenza virus A infection in airway epithelial cells activates the expression of eotaxin and that eotaxin may participate in the pathogenesis of airway inflammatory disease caused by viral infection, such as infectious type asthma .

Arch Ophthalmol, 2000 Jun, 118(6), 786 - 9
Disinfection of eyelid specula with chlorhexidine gluconate (Hibiclens) after examinations for retinopathy of prematurity; Hutchinson AK et al.; BACKGROUND: The preferred method of cleaning eyelid specula between examinations for retinopathy of prematurity is unknown . A previous study showed that disinfection with 70% isopropyl alcohol swabs fails to eliminate viruses and bacteria from the specula . OBJECTIVE: To determine if alternative sterilization procedures would allow multiple use of a single speculum without risking nosocomial infection . METHODS: In phase 1, 40 autoclave-sterilized eyelid specula were randomized into either "cleaned" or "patient control" groups after being used for routine retinopathy of prematurity examinations performed in the outpatient setting . Specula in the cleaned group were cleaned with chlorhexidine gluconate (Hibiclens) . Specula in the patient control group were not cleaned after use . All study specula were placed into enriched culture media from which bacterial and fungal cultures were obtained . In phase 2, 20 autoclave-sterilized eyelid specula were inoculated with a clinically relevant dilution of adenovirus serovar 5 or herpes simplex type 2 . Specula were randomized into either a cleaned or a control group, and cell cultures and immunofluorescence assays were used to document and confirm, respectively, viral growth . RESULTS: In phase 1, all 20 cultures from the patient control group grew bacteria compared with 0 (0%) of 20 cultures from the cleaned group and 0 (0%) of 5 from the cleaned control group . No fungi were isolated from any group . In phase 2, all 10 cultures from specula inoculated with adenovirus serovar 5 grew virus . None of the cultures from the 5 cleaned specula inoculated with herpes simplex type 2 grew virus . In contrast, all 5 cultures in the control group were positive for growth of herpes simplex type 2 . CONCLUSIONS: Autoclave sterilization is the ideal method of sterilization of eyelid specula between neonate examinations . When an alternative disinfection technique is required, washing the speculum with chlorhexidine gluconate and tap water is preferred over wiping with a 70% isopropyl alcohol swab . Arch Ophthalmol . 2000;118:786-789

Mol Reprod Dev, 2000 Jul, 56(3), 378 - 86
Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig; Kouba AJ et al.; Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy . To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured . Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry . To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured . Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts . To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12 . Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry . A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed . No difference could be detected for PAI-1 protein between breeds . The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined . Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12 . A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts . PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy . An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09) . Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA . Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo .

J Cell Biochem, 2000 Jun 6, 78(3), 371 - 9
ERK1/2 phosphorylation, induced by electromagnetic fields, diminishes during neoplastic transformation; Jin M et al.; It has been suggested that electromagnetic (EM) fields can act as co-promoters during neoplastic transformation . To examine this possibility, we studied the effects of 0.8-, 8-, 80-, and 300-microT 60-Hz electromagnetic (EM) fields in INITC3H/10T1/2 mouse fibroblast cells . These cells are transformed carcinogenically by methylcholanthrene, but the neoplastic phenotype can be suppressed indefinitely by the presence of retinyl acetate (RAC) in the culture medium . The effects of EM field exposures were examined at three stages: (1) before initiation of transformation (i.e., RAC in the culture media); (2) early in the transformation process (4 days after withdrawal of RAC); and (3) at full of neoplastic transformation (10 days after withdrawal of RAC) . EM field exposures induced significant increases in protein levels for hsp70 and c-Fos and in AP-1 binding activity . EM fields induced phosphorylation of MAPK/ERK1/2 before the onset of transformation, but these increases diminished during the transformation process . No phosphorylation in the other major extracellular stress pathway, SAPK/JNK, was detected in cells exposed to EM fields at any time before, during, or after neoplastic transformation . Human cells HL60, MCF7, and HTB124, exposed to EM fields, also showed MAPK/ERK1/2 phosphorylation . Cells treated with the phorbol ester, TPA, served as positive controls for AP-1 activation, c-Fos protein synthesis, and ERK1/2 phosphorylation . There was no indication that EM fields affected the rate of cell transformation or acted as a co-promoter, under the conditions of this study .

J Microbiol Methods, 2000 Jun, 41(1), 23 - 33
Adaptation of E . coli cell method for micro-scale nitrate measurement with the Griess reaction in culture media; Xu J et al.; The E . coli cell method for nitrate measurement consists of two-steps: nitrate reduction by the E . coli cell usually under anaerobic conditions and subsequently nitrite measurement with the Griess reaction . It was found that the E . coli DSM 498k wildtype cell can reduce nitrate to nitrite under aerobic conditions . Therefore, the E . coli method for nitrate measurement was adapted to be performed under aerobic conditions in a microtiter plate . The adapted method is simpler than the original E . coli method and other nitrate methods such as those with inorganic reductants and with purified enzymes . Furthermore, it was found that for the Griess reaction the pH values of samples after addition of the Griess reagent A should be lower than 1.8 for a stable absorbance at 540 nm to be reached . It is important to add the two Griess reagents separately and to read the absorbance twice consecutively in a microtiter plate . The adapted E . coli method was successfully applied to measure the traces of nitrate in MRS and other medium components by measuring the standard curve of a dilution of each individual medium component . It was found that many organic medium components contain traces of nitrate, while none of them contain detectable nitrite . Among these, the extract of meat and yeast extract contain relatively high amounts of nitrate: 217 mg N/kg and 99 mg N/kg respectively . MRS broth contains nitrate from 0.3 to 0.6 mg N/l depending on the batch numbers of the product . The adapted E . coli can also be used for nitrate measurement in other matrices.

Int J Antimicrob Agents, 2000 Jun, 15(1), 31 - 6
Quantitative RT-PCR analysis of multiple genes encoding putative metronidazole nitroreductases from Helicobacter pylori; Kwon DH et al.; Metronidazole (Mtz), a pro-drug, requires reductive activation by ferredoxin-like electron carrier proteins to kill bacteria and Mtz resistance is associated with a decrease or deficiency of Mtz nitroreductase activities in a target cell . Several genes encoding ferredoxin-like or -linked proteins such as pyruvate oxidoreductase (POR), ferredoxin oxidoreductase (FOR), ferredoxin (FdxA), ferredoxin-like protein (FdxB), flavodoxin (FldA) and oxygen insensitive nitroreductase (RdxA) have been identified from the complete genomic sequence of Helicobacter pylori . To understand the roles of these genes in H . pylori Mtz resistance, the gene expression for the proteins was examined using a method optimized for quantitative reverse transcription polymerase chain reaction (RT-PCR) . The RT-PCR products of FOR and RdxA were significantly decreased in the total RNA prepared from H . pylori cultured in the presence of Mtz as compared to the total RNA prepared from H . pylori cultured without Mtz in the media . A slight decrease, however, in band intensity of the RT-PCR products of the POR and, to a lesser extent, FdxB was obtained in the presence of Mtz . In contrast, the RT-PCR products of the FdxA, FldA, and GalE (UDP-galactose 4-epimerase; a control gene) were unchanged in total RNA prepared from H . pylori cultured with or without Mtz in the culture media . These results suggest that Mtz resistance may also be acquired by decreasing the transcription of some genes involved in Mtz reductive activation, in addition to the mutation in some individual genes such as rdxA.

Ann Otol Rhinol Laryngol, 2000 Jun, 109(6), 594 - 601
Secretory differentiation of serially passaged normal human nasal epithelial cells by retinoic acid: expression of mucin and lysozyme; Yoon JH et al.; The purpose of this study was to subculture normal human nasal epithelial (NHNE) cells without compromising their ability to differentiate into secretory and ciliated cells and to study the effect of retinoic acid on mucous and serous secretions in passaged cells and to compare the expression of mucin and lysozyme in cultured cells with those in in vivo nasal epithelium . The subcultured cells were tested after every passage for secretory differentiation in air-liquid interface cultures . The cultured NHNE cells secreted mucin and lysozyme . The cells became squamous and mucin secretion decreased when retinoic acid was deleted from the culture media . Cells from passage 1 through passage 2 remained able to differentiate into mucous or squamous cells . Mucin gene 4 (MUC4), MUC5AC, MUC7, MUC8, and lysozyme messenger RNAs were expressed in passage 2 NHNE cells . In conclusion, passage 2 NHNE cell cultures retain features of normal epithelium and are suitable for many studies of upper airway cell biology.

J Hepatol, 2000 May, 32(5), 762 - 70
A p160ROCK-specific inhibitor, Y-27632, attenuates rat hepatic stellate cell growth; Iwamoto H et al.; BACKGROUND/AIMS: p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers . Recently, Rho signaling pathways were reported to play an important role in the activation of rat hepatic stellate cells (HSC) . The aim of this study was to investigate the mechanism of action of a p160ROCK-specific inhibitor, Y-27632, on cultured rat HSC . METHODS: HSC were isolated from normal rat livers and cultured on fibronectin-coated dishes . The cell morphology and actin cytoskeleton were studied with phase contrast and fluorescence microscopy, respectively . Immunoblot analysis was used to examine phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, and the expression of cell cycle-associated proteins . HSC proliferation was measured by quantitating the percentage of cells that exhibited nuclear incorporation of 5-bromodeoxyuridine . Type I collagen gene expression and accumulation in HSC culture media were evaluated by Northern blot and enzyme-linked immunosorbent assay, respectively . RESULTS: Y-27632 consistently blocked cell spreading and suppressed RhoA-induced formation of stress fibers in HSC . In addition, Y-27632 inhibited phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase . Cells treated with Y-27632 failed to proliferate, in contrast to untreated spread cells . This shape-dependent block in cell proliferation correlated with a failure to increase cyclin D1 protein level and to down-regulate the cell cycle inhibitor p27 . Y-27632 decreased type I collagen gene expression and accumulation in HSC culture media . CONCLUSIONS: Our findings indicate that p160ROCK-mediated actin stress fiber assembly is involved in the pathophysiology of hepatic fibrogenesis and suggest that inhibitors of the RhoA-ROCK pathway might be useful therapeutically in liver fibrogenesis.

Gene Ther, 2000 Jun, 7(11), 910 - 3
Production and concentration of pseudotyped HIV-1-based gene transfer vectors; Reiser J; Strategies to generate highly concentrated HIV-1 vector pseudotypes involving different envelope (Env) proteins including the vesicular stomatitis virus (VSV) G glycoprotein, the Moloney murine leukemia virus (MLV) 4070A amphotropic Env and the rabies G glycoprotein were established . Virus stocks were prepared by transient transfection using standard cell culture media or serum-free media . Such stocks were concentrated 50- to 300-fold by ultracentrifugation or by ultrafiltration using Centricon Plus-80 units yielding titers of up to 109transducing units per milliliter . There was no loss in titer with any of the pseudotypes tested . Thus, like lentiviral vectors pseudotyped with VSV-G, HIV-1-based vectors pseudotyped with the MLV 4070A amphotropic Env and the rabies G glycoprotein resist inactivation during concentration . This opens up the possibility to generate highly concentrated HIV-1 vector stocks carrying alternative Env proteins on a large scale.

J Bone Miner Res, 2000 Jun, 15(6), 1189 - 97
Molecular events that contribute to lysyl oxidase enzyme activity and insoluble collagen accumulation in osteosarcoma cell clones; Uzel MI et al.; Maximum collagen synthesis and maximum accumulation of insoluble collagen occur at different phenotypic stages in developing osteoblastic cell cultures . Insoluble collagen accumulation depends in part on the activity of extracellular enzymes including procollagen N-proteinases, procollagen C-proteinase (derived from the BMP1 gene), and lysyl oxidase . In addition to its action on procollagen, procollagen C-proteinase processes prolysyl oxidase to mature 32-kDa lysyl oxidase . The regulation of extracellular activities that control insoluble collagen accumulation has not been studied extensively . The present study compares molecular events that control production of a collagenous mineralized extracellular matrix in vitro among five different murine osteosarcoma cell clones derived from the same tumor, but which differ in their ability to produce an insoluble mineralized matrix . Levels of insoluble type I collagen, insoluble calcium, bone morphogenetic protein 1 (BMP-1), and lysyl oxidase expression, lysyl oxidase biosynthesis, lysyl oxidase activity, and prolysyl oxidase processing activity were determined . Results surprisingly indicate that lysyl oxidase activity is not related closely to lysyl oxidase messenger RNA (mRNA) levels among the different cell clones . However, it appears that BMP-1-dependent prolysyl oxidase processing could contribute to the observed lysyl oxidase activity . Highest collagen and BMP-1 mRNA levels, prolysyl oxidase processing activity, and lysyl oxidase activity occurred in a cell clone (K8) that showed the highest levels of insoluble collagen accumulation . Culture media from a cell clone (K37) that accumulates little insoluble collagen or calcium but expresses high levels of lysyl oxidase mRNA contained low molecular weight fragments of lysyl oxidase protein and showed low lysyl oxidase activity . By contrast the K14 cell line exhibits relatively high lysyl oxidase activity and collagen accumulation, but low levels of mature lysyl oxidase protein . Together, these studies indicate that catabolic as well as anabolic activities are important in regulating insoluble collagen accumulation in osteoblastic cells . In addition, results suggest that products of genes homologous to lysyl oxidase may contribute to observed lysyl oxidase activity.

Biochim Biophys Acta, 2000 Jun 2, 1497(1), 127 - 34
Oversulfated fucoidan inhibits the basic fibroblast growth factor-induced tube formation by human umbilical vein endothelial cells: its possible mechanism of action; Soeda S et al.; We have previously demonstrated that chemically oversulfated fucoidan (OSF) but not native fucoidan (NF) effectively suppresses the tube structure formation by human umbilical vein endothelial cells (HUVEC) on the basement membrane preparation, Matrigel . In this study, using more defined systems where basic fibroblast growth factor (bFGF) induces the tube formation by HUVEC on collagen gel, we investigated the mechanism responsible for the inhibition of angiogenesis by OSF in vitro . Unlike NF and desulfated fucoidan (desF), OSF potently inhibited the bFGF-induced HUVEC migration and tube formation . ELISA for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the culture media indicated that OSF increased the bFGF-induced release of PAI-1 antigen, but not of t-PA antigen . Analyses of the binding of bFGF to HUVEC surfaces and the following protein tyrosine phosphorylation revealed that OSF could promote the cell binding and autophosphorylation of 140 and 160 kDa receptors . In heparitinase-treated HUVEC, contrarily, the bFGF binding and PAI-1 release were decreased by OSF . These results suggest that OSF is a highly sulfated unique polysaccharide that can promote the binding of bFGF to the heparan sulfate molecules required for binding to the high affinity receptors with tyrosine kinase activity . The resultant increase in PAI-1 release may play a key role for the prevention of cell migration accompanied by matrix proteolysis.

J Cardiovasc Pharmacol, 2000 Jun, 35(6), 887 - 90
Amlodipine inhibits expression of matrix metalloproteinase-1 and its inhibitor in human vascular endothelial cells; Ikeda U et al.; Matrix metalloproteinase-1 (MMP-1) may play an important role in the pathogenesis of atherosclerosis and atherosclerotic plaque rupture . We investigated the effect of the calcium channel blockers amlodipine and nifedipine on the expression of MMP-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in endothelial cells (ECs) . MMP-1 and TIMP-1 levels in conditioned media of human vascular ECs were measured by enzyme-linked immunosorbent assay . Collagenolytic activity was determined by fluorescence-labeled collagen digestion . The addition of interleukin-1beta (IL-1beta) increased MMP-1 levels in the culture media of ECs . Amlodipine, but not nifedipine, significantly decreased MMP-1 levels in IL-1beta-stimulated ECs . TIMP-1 levels also were significantly increased by IL-1beta, and its expression was slightly decreased by amlodipine, not by nifedipine . Amlodipine significantly inhibited collagenolytic activity in the culture media of IL-1beta-stimulated ECs, whereas nifedipine showed no significant effect on the activity . Our findings revealed that amlodipine, but not nifedipine, inhibits IL-1beta-induced MMP-1 expression in human ECs.

Rev Med Chil, 1999 Nov, 127(11), 1305 - 11
{In vitro immunosuppressive effect of low density lipoproteins}; Gonzalez M et al.; BACKGROUND: Immune cells participate in the formation of atheromatous plate, however little is known about the effects of native or oxidatively modified lipoproteins on these cells . AIM: To study the effects of lipoproteins on in vitro mononuclear cell proliferation . MATERIAL AND METHODS: Peripheral blood mononuclear cells were obtained from 10 patients with type 2 diabetes mellitus (aged 52 +/- 9 years old with a disease duration of 8.2 +/- 5.7 years and a mean glycosilated hemoglobin of 9.3 +/- 2.2%) and 10 non diabetic healthy controls (aged 50.3 +/- 7.1 years old) . These were stimulated with phytohemagglutinin (PHA) alone or in the presence of native LDLS, malondialdehyde modified LDLs or glycated LDLs . Proliferation was measured as 3H-thymidine incorporation and expressed as Stimulation Index (SI) . RESULTS: SI of patients and healthy subjects, after PHA stimulation were similar: (57.5 +/- 29.8 and 61.1 +/- 23.5) respectively LDLs did not induce proliferation in neither group . Native LDLs produced a 98% inhibition of PHA induced proliferation . Malondialdehyde modified and glycated LDLs caused a 50% inhibition . The suppressive effect was maintained when lipoproteins were incorporated to culture media 60 min prior or after PHA stimulation . CONCLUSIONS: Lipoproteins inhibit in vitro PHA induced peripheral blood mononuclear cell proliferation both in diabetic and in non diabetic subjects.

Placenta, 2000 May, 21(4), 354 - 60
Production of interleukin (IL)-6 and IL-8 by a choriocarcinoma cell line, BeWo; Fujisawa K et al.; To clarify the biological and pathological features of choriocarcinoma, we evaluated the in vitro production of cytokines by a choriocarcinoma cell line, BeWo . We measured the concentration of interleukin (IL)-6 and IL-8 in the culture media of BeWo cells after stimulation with various modulatory agents of cytokine expression by enzyme-linked immunosorbent assays . Northern blot analysis was used to examine the expression of IL-6 and IL-8 mRNA in these cells . A weak expression of IL-6 and IL-8 was detected in unstimulated BeWo cells by both methods . IL-6 transcription and secretion were dose-dependently enhanced by stimulation with IL-1beta, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1 and 12-O-tetradecanoyl phorbol-13-acetate (TPA) . Forskolin, lipopolysaccharide and interferon-gamma had no effect on these cytokines production . The TNF-alpha-induced secretion of IL-6 was inhibited by dexamethasone . The TPA-induced production of IL-6 was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and sphyngosine, suggesting the involvement of a protein kinase C-dependent pathway . Levels of IL-8 mRNA and protein were also dose-dependently increased by stimulation with IL-1beta, TNF-alpha and TPA . In contrast to IL-6, the expression of IL-8 was not affected by TGF-beta1 . It is suggested that, in addition to the production of steroidal and non-steroidal hormones, these cytokines may serve as part of a cytokine network that modulates the proliferation and angiogenesis of choriocarcinomas .

J Endocrinol, 2000 Jun, 165(3), 617 - 24
Expression of vascular endothelial growth factor in response to high glucose in rat mesangial cells; Kim NH et al.; Diabetic nephropathy associated with hyperglycemia is characterized by glomerular hyperfiltration and endothelial dysfunction . Vascular endothelial growth factor (VEGF) is known to be primarily involved in neoangiogenesis and increased endothelial permeability . The purpose of this study was to investigate VEGF expression in response to high glucose in rat cultured mesangial cells and to identify its signal pathway via protein kinase C (PKC) . Rat mesangial cells were cultured with different concentrations of glucose: normal (5 mM d-glucose), medium (15 mM d-glucose) and high (30 mm d-glucose) . Calphostin-C as a PKC inhibitor and phorbol myristate acetate (PMA) as a PKC downregulator were instillated into culture media to evaluate the role of PKC in mediating the glucose-induced increase in VEGF expression . High glucose increased expression of VEGF at the mRNA and protein levels, identified by semi-quantitative RT-PCR and western blotting, within 3 h and in a time- and glucose concentration-dependent manner . Calphostin-C and PMA inhibited glucose-induced increases in VEGF expression at the mRNA and protein levels . In conclusion, high glucose can directly increase VEGF expression in rat mesangial cells via a PKC-dependent mechanism . These results suggest that VEGF could be a potential mediator of glomerular hyperfiltration and proteinuria in diabetic nephropathy.

Hum Reprod, 2000 Jun, 15(6), 1250 - 5
Elevated expression of tumour necrosis factor alpha in cultured granulosa cells from women with endometriosis; Carlberg M et al.; Fertilization and oocyte cleavage rates have previously been demonstrated to be lower for women with endometriosis undergoing IVF compared with controls . This might be related to impaired oocyte function, possibly due to an inflammatory milieu in the pelvis of these women, where an elevated concentration of many cytokines is documented . The aim of this study was to examine whether granulosa cells from women with endometriosis deviated with respect to production of the inflammatory cytokines interleukin-1beta, interleukin-6, interleukin-8 and tumour necrosis factor alpha (TNFalpha) compared with granulosa cells from healthy women, undergoing IVF for male infertility . The effect of human chorionic gonadotrophin on cytokine production was also investigated . Granulosa cells in follicular fluid were obtained at oocyte retrieval for IVF . Incubated cell culture media were analysed by enzyme-linked immunosorbent assay . The basal production of all four cytokines was higher in cells from women with endometriosis when compared to controls, although the increase was only significant for TNFalpha . Chorionic gonadotrophin had no significant effect, although it had a tendency to suppress cytokine release in both patient categories . Whether aberrant cytokine production in granulosa cells from women with endometriosis may disturb fertilizing capacity of oocytes requires study.

Free Radic Res, 2000 Jul, 33(1), 45 - 56
Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death; Miwa H et al.; Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death . Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci . Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined . Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner . H2O2 but not O2*- produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H2O2 as ROS stress thereafter . The H2O2-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2-3 days for LCL, Raji and BJAB cells . The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines . Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells . alpha-Cyano-4-hydroxycinnamate, a specific inhibitor of the H+-monocarbohydrate transporter, increased the H2O2-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media . These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.

Mol Hum Reprod, 2000 Jun, 6(6), 541 - 8
The effect of mifepristone administration on leukocyte populations, matrix metalloproteinases and inflammatory mediators in the first trimester cervix; Denison FC et al.; Cervical ripening is analogous to an inflammatory reaction characterized by an influx of inflammatory cells and an increase in inflammatory mediators . The anti-gestogen mifepristone is highly effective in inducing cervical ripening in women throughout gestation . However, its mechanism of action is largely unknown . The aim of the study was to investigate the effect of in-vivo administration of mifepristone on inflammatory cells and mediators in the cervix . Cervical biopsies were taken from women undergoing a first trimester termination of pregnancy at 0, 6, 12, 24 and 36 h (n = 6 per group) after mifepristone administration . Biopsies were fixed for immunohistochemistry and also cultured for subsequent analysis of culture media by radioimmunoassay or enzyme-linked immunosorbent assay . After administration of mifepristone (6-24 h), there was an increase in immunostaining for leukocyte common antigen (CD45), neutrophil elastase, monocytes (CD68), and matrix metalloproteinases (MMP)-1, -8 and -9 . Immunostaining for MMP-2 and tissue inhibitor of metalloproteinases (TIMP)-1, -2 and -4 were unaffected by mifepristone treatment . Secretion of monocyte chemotactic protein (MCP-1) was significantly (P < 0.05) increased from biopsies taken 6-24 h after mifepristone administration . Cervical biopsies also released interleukin-8 (IL-8), prostaglandin (PG) E(2), PGF(2alpha) and prostaglandin metabolites (PGEM and PGFM) although their secretion was unaffected by mifepristone treatment . This study suggests that mifepristone may, in part, effect cervical ripening by modulating the influx of inflammatory cells into the cervix, up-regulating MMP expression and inducing chemokine secretion by cervical tissue.

J Biol Chem, 2000 Aug 11, 275(32), 24429 - 35
Development and characterization of a recombinant truncated type VII collagen "minigene" . Implication for gene therapy of dystrophic epidermolysis bullosa; Chen M et al.; Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder of skin caused by mutations in the type VII collagen gene . The lack of therapy for DEB provides an impetus to develop gene therapy strategies . However, the full-length 9-kilobase type VII collagen cDNA exceeds the cloning capacity of current viral delivery vectors . In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous domains, NC1 and NC2, and part of the central collagenous domain using stably transfected human 293 cell clones and purified large quantities of the recombinant minicollagen VII from culture media . Minicollagen VII was secreted as correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation . Purified minicollagen VII bound to fibronectin, laminin-5, type I collagen, and type IV collagen . Furthermore, retroviral-mediated transduction of the minigene construct into DEB keratinocytes (in which type VII collagen was absent) resulted in persistent synthesis and secretion of a 230-kDa recombinant minicollagen VII . In comparison with parent DEB keratinocytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-substratum adhesion, increased proliferative potential, and reduced cell motility, features that reversed the DEB phenotype toward normal . We conclude that the use of the minicollagen VII may provide a strategy to correct the cellular manifestations of gene defects in DEB.

Ann N Y Acad Sci, 2000, 900, 325 - 35
Biological factors in culture media affecting in vitro fertilization, preimplantation embryo development, and implantation; Loutradis D et al.; Optimal culture conditions are of paramount importance for in vitro fertilization of gametes, preimplantation embryo development, and implantation for all species . Water is the basis of all culture media, and ultrapure water should be employed . The main energy sources of a medium are lactate, pyruvate, and glucose . The concentrations of the first two vary in different media, whereas the latter is necessary mainly for the later stages (morula to blastocyst) of development . A fixed nitrogen source is essential for implantation embryo development whether this is provided by amino acids, albumin, or serum . Suboptimal culture conditions can block development . Pronuclear zygotes of most species (but not human) arrest at some point between the two-cell and the 16-cell stage . Modifying culture conditions can lead the embryos to develop through this block . Hypoxanthine also causes a two-cell block to mouse pronuclear zygotes, and this again depends largely on culture conditions . Simple culture media are bicarbonate-buffered systems with pyruvate, lactate, and glucose . Complex media, such as Ham's F-10, contain in addition amino acids and other elements found in serum . Human tubal fluid simulates the fallopian tube microenvironment . EDTA, gonadotropins, growth factors, and other substances can be included in the media to stimulate development . Coculture of embryos with oviductal cells has shown promising results.

Neuroreport, 2000 Apr 27, 11(6), 1357 - 60
BN 80933 inhibits F2-isoprostane elevation in focal cerebral ischaemia and hypoxic neuronal cultures; Marin JG et al.; Formation of the lipid peroxidation product 8-epi-prostaglandin2alpha (8-epi-PGF2alpha) a bioactive marker of oxidative stress, was quantified in in vitro and in vivo models of neuronal death . In culture media of primary rat cortical neurones exposed to hypoxia followed by reoxygenation, a 3.7-fold increase of 8-epi-PGF2alpha concentration was observed in comparison to control cells . In rats submitted to 2h middle cerebral artery occlusion followed by a 22h reperfusion period, a 27-fold increase of 8-epi-PGF2alpha was observed in the ischaemic hemisphere compared with the corresponding hemisphere of sham-operated rats . Treatment with the neuroprotective agent BN 80933 significantly reduced both 8-epi-PGF2alpha elevations in vitro and in vivo . These data suggest that 8-epi-PGF2alpha elevations might reflect the damaging free radical overproduction and subsequent lipid peroxidation during neuronal injury induced by hypoxia and ischaemia . Inhibition of 8-epi-PGF2alpha elevations participates to the neuroprotective effects of BN 80933.

Lakartidningen, 2000 Apr 5, 97(14), 1660 - 2, 1665-6
{The interplay between embryo and endometrium is important for successful implantation . Increased knowledge can result in new contraceptive methods and better treatment of infertility}; Danielsson KG; It seems likely that local factors such as cytokines play an important role in synchronizing the development of the embryo and the gestational tract and in determining pregnancy outcome . Evaluation of the precise sequence of cytokines to which the embryo is exposed and responds during the critical periimplantation period may also make it possible to develop culture media which will improve embryo viability and hence increase implantation rates in IVF programs . In addition, an in vitro three-dimensional culture system is being developed which closely resembles endometrium in vivo . This system offers an opportunity to study the local molecular events involved in blastocyst adhesion and to elucidate the role played by local factors, such as cytokines, in producing a receptive luminal epithelium.

Int J Mol Med, 2000 Jun, 5(6), 651 - 6
Influence of serum from healthy or breast tumor-bearing women on the growth of MCF-7 human breast cancer cells; Cos S et al.; Sera from women healthy (HW) or with breast (BCW), ovarian or endometrial cancer, were added (10%) to the culture media of MCF-7 cells and cell proliferation assessed 4 days later to verify: a) whether sera from BCW, obtained before or 8 days after tumor ablaction, influence the proliferation of these cells, b) whether the effects of serum from BCW are specific for mammary tumor cells . Sera from BCW, but not sera from women with ovarian or endometrial cancer, increased MCF-7 cell proliferation in comparison with sera from HW . After surgical ablation of the breast tumors, serum's ability to increase MCF-7 cell proliferation decreased significantly . These effects cannot be explained by differences on serum levels of estradiol or melatonin . These results suggest the presence of growth-promoting substances of possible tumoral origin in serum of BCW, a fact that should be considered as support for the surgical treatment of tumor masses.

J Gen Virol, 2000 Jun, 81(Pt 6), 1545 - 52
Characterization of interaction of gH and gL glycoproteins of varicella-zoster virus: their processing and trafficking; Maresova L et al.; Varicella-zoster virus (VZV) glycoproteins gH and gL were examined in a recombinant vaccinia virus system . Single expression of glycoprotein gL produced two molecular forms: an 18 kDa form and a 19 kDa form differing in size by one endoglycosidase H-sensitive N-linked oligosaccharide . Coexpression of gL and gH resulted in binding of the 18 kDa gL form with the mature form of gH, while the 19 kDa gL form remained uncomplexed . The glycosylation processing of gL was not dependent on gH; however, gL was required for the conversion of precursor gH (97 kDa) to mature gH (118 kDa) . Subsequent analyses indicated that gL (18 kDa) was a more completely processed gL (19 kDa) . Screening of the culture media revealed that gH and gL were secreted, but only if coexpressed and complexed together . The secreted form of gL was 18 kDa while that of gH was 114 kDa . The fact that secreted gH was smaller than intracytoplasmic gH suggested a proteolytic processing event prior to secretion . The 19 kDa form of gL was never secreted . These findings support a VZV gL recycling pathway between the endoplasmic reticulum and the cis-Golgi apparatus.

Cell Tissue Res, 2000 Apr, 300(1), 11 - 9
Ependymal explants from the lateral ventricle of the adult bovine brain: a model system for morphological and functional studies of the ependyma; Perez-Martin M et al.; By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells . Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days . For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP) . For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ . The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP . The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum . In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP . These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells . The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma . Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.

J Bone Miner Res, 2000 May, 15(5), 894 - 901
Thiazide diuretics affect osteocalcin production in human osteoblasts at the transcription level without affecting vitamin D3 receptors; Lajeunesse D et al.; Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density . Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown . We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63 . HCTZ dose-dependently (1-100 microM) inhibited 1,25-dihydroxyvitamin D3 {1,25(OH)2D3}-induced OC release by these cells (maximal effect, -40-50% and p < 0.005 by analysis of variance {ANOVA}) as measured by ELISA . This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (-47.2+/-4.0%; p < 0.0001 by paired ANOVA with 100 microM 13.6+/-0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells . Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, -22.5+/-6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion . Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels . Both Northern and Western blot analyses showed no effect of HCTZ (1-100 microM) on VDR levels . The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ . The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS . Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media . In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA . These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels . As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate.

J Vet Med A Physiol Pathol Clin Med, 2000 Mar, 47(2), 99 - 105
Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses; Okumura M et al.; The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage . Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3) . Chondrocytes were centrifuged and cultured as pellets . Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks . The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-linked immunosorbent assay using a 1/20/5D4 antibody, respectively . The concentration of GAG was significantly increased both in the media of pellets stimulated by IGF-I alpha and in those stimulated by IL-1 alpha . KS concentration was significantly increased in those stimulated by IL-1 alpha, while no significant change was found in those stimulated by IGF-I alpha . A high correlation between GAG and KS concentrations was found in the media of pellets stimulated by IL-1 alpha (r = 0.84), but not in those stimulated by IGF-I alpha (r = 0.59) . The results suggest that the concentration of KS reacting to 1/20/5D4 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage . The KS concentration in biological fluids could therefore be a useful marker to understand further the cartilage catabolic process . It may also represent some aspects of the cartilage anabolic process.

Diabetes Res Clin Pract, 2000 May, 48(2), 127 - 38
IDL can stimulate atherogenic gene expression in cultured human vascular endothelial cells; Maeno Y et al.; Previously, we have reported that the lipoprotein fraction containing intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) isolated from diabetics stimulates an atherogenic cytokine in cultured endothelial cells . To study which lipoprotein fraction isolated from diabetics can modulate the gene expression in endothelial cells, we isolated IDL and LDL fractions from 14 type 2 diabetics and seven age- and BMI- adjusted non-diabetics . We measured the effects of the lipoproteins on mRNA expression of atherogenic molecules in cultured endothelial cells . We found that the IDL fraction stimulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression in endothelial cells as time- and dose-dependent fashions, while the LDL fraction was not effective . IDL isolated from diabetics also increased not only platelet-derived growth factor B-chain, but also intercellular adhesion molecule-1 mRNA contents . Furthermore, the HbA(1c) levels in diabetics were significantly correlated with their abilities of IDL to increase MCP-1 mRNA content in the cells and the increment coincided with the increase in MCP-1 protein release into culture media . These results indicate that qualitative as well as quantitative changes in IDL fraction in diabetes are atherogenic through stimulating gene expression of atherogenic molecules in endothelial cells.

Cancer Lett, 2000 Jun 1, 154(1), 53 - 62
Intercellular communication and cell proliferation in precision-cut rat liver slices: effect of medium composition and DDT; de Graaf IA et al.; Gap junctional intercellular communication (GJIC) and cell proliferation were studied in control and 1,1'-bis(p-chlorophenyl)-2, 2,2,-trichloroethane (DDT) treated precision-cut liver slices of rat by evaluating connexin 32 (Cx32) expression and 5-bromo-2'-deoxyuridine (BrdU) incorporation . In addition, the effect of different culture media (RPMI and WME) on control and DDT influenced Cx32 expression and cell proliferation was determined . Cx32 expression in control precision-cut liver slices was maintained during 8 h of culturing, but decreased after prolonged culturing . Control cell proliferation was significantly higher when WME was used as culture medium than when RPMI was used . In slices treated with DDT Cx32 expression was decreased . In slices cultured in RPMI medium, this decrease preceded a dose-dependent increase in cell proliferation . These results show the usefulness of precision-cut liver slices in studying cellular proliferation and intercellular communication.

J Clin Microbiol, 2000 May, 38(5), 1984 - 7
Growth supplements for Helicobacter pylori; Jiang X et al.; The growth response of Helicobacter pylori in broth was determined in the presence of ferrous sulfate, sodium pyruvate, and mucin (porcine stomach) . The addition of either ferrous sulfate and sodium pyruvate or mucin to brain heart infusion broth with 7% horse serum (BHI-HS) enhanced the growth of H . pylori . The best growth of strain NB2-1, which was the slowest growing of 10 H . pylori strains evaluated, occurred in the presence of 0.05% ferrous sulfate and 0.05% sodium pyruvate . The addition of 0.3% mucin to BHI-HS reduced the lag time of H . pylori by 48 h and enhanced the growth . On the basis of the results for 10 H . pylori strains, the combination of ferrous sulfate (0.025%), sodium pyruvate (0.025%), and mucin (0.15%) in BHI-HS counteracted the inhibitory effects of the antibiotics used in culture media for selective growth of H . pylori . Results suggest that these supplements may be useful for enhancement of the growth of H . pylori in enrichment media.

Br J Cancer, 2000 May, 82(9), 1553 - 6
The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation; Butler SA et al.; Ectopic production of free beta human chorionic gonadotrophin (hCGbeta) by bladder carcinoma is well described and occurs in approximately 35% of cases . hCGbeta secreting tumours are more aggressive, radioresistant and have a greater propensity to metastasize . We proposed that the ectopic production of hCGbeta was contributing in an autocrine fashion to the radioresistance and metastatic potential of such secreting tumours . Though we demonstrated that the addition of hCGbeta to the culture media of bladder, cervical and endometrial carcinoma cell lines brought about an increase in cell populations this was not accompanied by a significant increase in the rate of replication . Since a cell population size is a balance of mitosis and mortality, we proposed that hCGbeta was inhibiting apoptosis . Here we have demonstrated that following incubation with recombinant hCGbeta, bladder carcinoma cells refrain from undergoing apoptosis . Quantitation of apoptotic bodies was carried out by immunoassay and corrected to cell number as determined by MTT assay . In each cell line, addition of hCGbeta reduced the number of apoptotic bodies dose-dependently, indicating a diminished apoptotic rate . Furthermore, TGFbeta1-induced apoptosis could be dose-dependently inhibited by co-incubation with hCGbeta . We propose, therefore, that such a decline in apoptosis may account for the cell population increase previously reported . It may also explain the radioresistance and aggressive nature of hCGbeta-secreting tumours and the poor prognosis associated therein.

J Biochem Mol Toxicol, 2000, 14(4), 204 - 9
Induction of CYP1A by carbofuran in primary culture of fish hepatocytes; Ghosh MC et al.; Carbofuran is a nematicide used in agricultural fields throughout the world . Indiscriminate use of this pesticide poses severe detrimental effects on our ecosystem . We have shown that it induces the CYP1A (cytochrome P4501A) monooxygenase enzyme system in cultured hepatocytes from Indian catfish, Heteropneustes fossilis (Bloch) . We have quantified this induction by measuring the activity of the enzyme 7-ethoxyresorufin-O-deethylase (EROD), synthesized from CYP1A1 gene . The induction followed a dose-dependent relationship with carbofuran . The dose-dependent curve of EROD using carbofuran was very much similar with beta-napthoflavone, which is a known inducer of CYP1A1 . Coexposure of these compounds to the culture media showed a synergistic effect on the enzyme activity . A blocker of aromatic hydrocarbon receptor, alpha-napthoflavone, blocked carbofuran-induced EROD activity in a dose-dependent manner . All these findings suggest that metabolism of carbofuran might be mediated by the CYP1A monooxygenase system through binding of the aromatic hydrocarbon receptor . We have also studied the superinduction phenomenon, which is a typical characteristic of the CYP1A gene in our system.

Toxicology, 2000 Apr 3, 144(1-3), 57 - 61
Effect of selenium supplementation on the activities of glutathione metabolizing enzymes in human hepatoma Hep G2 cell line; Helmy MH et al.; Cell culture is an important tool for studying injury to cells exposed to oxidative stress . The human hepatoblastoma derived Hep G2 cells retain their morphology and most of their function in culture and are therefore widely used as an in vitro model of human hepatocytes . Conventional cell culture media are deficient in selenium, which is essential for activation of glutathione peroxidase (GPx), a key enzyme in the defense against oxidative stress . Supplementation of the culture media with 1 microM sodium selenite increased the activities of total GPx by threefold and the selenium-dependent GPx by fourfold as compared to cells cultured in control media . The non-selenium-dependent GPx activity was unchanged . The activities of the other glutathione (GSH)-related enzymes were practically unchanged despite a tendency toward elevation . The activities of oxidized glutathione (GSSG) reductase and catalase increased by 22.4 and 27.4%, respectively . These relatively small increases did not carry statistical significance . Supplementation of tissue culture media with selenium may prove important, particularly for cell protection against oxidative stress.

Atherosclerosis, 2000 May, 150(1), 63 - 70
Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression; Hojo Y et al.; The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs) . Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured . VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay . Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe . Immunohistochemistry was performed to determine which types of cell produce VEGF . Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively . Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h . Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF . Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production . Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs . A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell . An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures . Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.

J Vet Med B Infect Dis Vet Public Health, 2000 Feb, 47(1), 1 - 8
The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics; Alonso-Villalobos P et al.; The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media . AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA) . In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion . Acetylthiocholine iodide was the favoured substrate of the enzyme . When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed . The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition . Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated . The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT) . The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.

Ontogenez, 2000 Mar-Apr, 31(2), 139 - 43
{The effect of the composition of the culture media on bovine oocyte maturation and embryo development in vitro}; Smetanina IG et al.; We studied the capacity of the cattle oocyte for the resumption of meiosis and achievement of metaphase II in various protein-free culture media (DMEM, TCM-199, Ham's F-10, and Ham's F-12) and the pattern of influence of the estrous serum on in vitro development of fertilized cattle oocytes, with special reference to the time of its addition to the synthetic oviduct fluid containing BSA . In the first experimental series, it was shown that the highest number of oocytes (76.1%) resumed meiosis in DMEM medium . Meiosis was not resumed in Ham's F-12 . Intermediate results were obtained for TCM-199 (55.1%), which is commonly used for maturation of cattle oocytes in vitro, and for Ham's F-10 (51.7%) . The oocytes reached metaphase II in DMEM at a higher rate (45.3%) than in TCM-199 or Ham's F-10 (29.4 and 8.6%, respectively) . In the second experimental series, the estrous serum was added to the culture medium within 20 h (control) or 42 h (experiment) after the beginning of fertilization . The estrous serum did not inhibit the first cleavage division (the percentage of cleaving embryos did not differ reliably: 32.7 and 37.9%, respectively) . However, a later serum addition to the culture medium (within 42 h after the beginning of fertilization) reliably increased the percentage of embryos that reached the blastocyst stage (6.5% in the control and 17.8% in the experiment) and the hatched blastocyst stage (2 and 9.2%, respectively).

J Am Acad Dermatol, 2000 May, 42(5 Pt 1), 841 - 4
Antiepiligrin cicatricial pemphigoid; Hsu RC et al.; Cicatricial pemphigoid is a chronic subepithelial autoimmune blistering disease of mucous membranes and skin . Recently, a subtype of cicatricial pemphigoid with autoantibodies to epiligrin was identified . We describe a Taiwanese patient who presented with ocular, oral, and cutaneous involvement . Direct immunofluorescence showed IgG and C3 deposition in epidermal basement membrane; indirect immunofluorescence showed circulating IgG autoantibodies reactive with the dermal side of 1 mol/L sodium chloride-split skin . Immunoblotting of laminin 5 isolated from the extracellular matrix of cultured human keratinocytes showed no specific reactivity . In contrast, with immunoprecipitation of the conditioned culture media from biosynthetically radiolabeled human keratinocytes, this patient's serum clearly reacted with a series of disulfide-linked polypeptides that correspond to laminin 5(alpha3beta3gamma2) and laminin 6(alpha3beta1gamma1) . This is the first confirmed case of a patient of Chinese ancestry with this disease entity.

Vet J, 2000 May, 159(3), 282 - 6
Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol; Otoi T et al.; Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME) . After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol . The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture . The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts . These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate .

Biol Reprod, 2000 May, 62(5), 1459 - 65
Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro; Crosier AE et al.; The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis . Compact morulae produced in vivo were obtained from superovulated Holstein cows . Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows . The complexes were matured and fertilized in vitro . At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA . At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy . The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula . The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo . There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05) . For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05) . The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo . In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo . Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.

Intervirology, 2000, 43(1), 1 - 5
Effects of varying concentrations of bleach on in vitro HIV-1 replication and the relevance to injection drug use; Contoreggi C et al.; The use of bleach (hypochlorite) as a disinfectant for drug injection equipment in the intravenous-drug-using population was recommended early in the HIV-1/AIDS epidemic . Epidemiological studies have challenged the use of bleach as an effective measure to prevent HIV-1 transmission . However, in vitro HIV-1 coculture studies have shown that a high concentration of bleach is an effective cytotoxic and potentially virucidal agent . In this study, we demonstrate that HIV-1 peripheral blood mononuclear cell cocultures containing low concentrations of hypochlorite in the media showed earlier conversion to HIV-1 positivity, as measured by the presence of p24 antigen . HIV-1 cocultures with high concentrations of hypochlorite in the culture media, which appeared to be highly cytotoxic, and HIV-1 cocultures without bleach in the media did not exhibit this early p24 antigen positivity . Hypochlorite chemically disinfects by releasing free chlorine that is a potent oxidant . In injection drug equipment, a low residual concentration of bleach is likely to remain in cleaned equipment despite rinsing with water . Low concentrations of oxidants have been shown to enhance tissue inflammation, in vivo, as well as HIV-1 replication in vitro . Previous studies have shown that despite vigorous cleaning of blood-contaminated injection syringes with bleach followed by water, microaggregates of residual blood remained in bleach-cleaned blood-contaminated syringes . Hypothetically, oxidant effects of the residual bleach in the bleach-cleaned syringes could enhance the possibility of infection by remaining HIV-1 contained in a contaminated syringe . We suggest that the likelihood of an injection drug user contracting HIV-1 through the sharing of a bleach-cleaned blood-contaminated syringe may be increased by the cotransmission of residual bleach and its localized tissue-inflammatory effects; however, this has not been statistically proven in epidemiological studies .

Connect Tissue Res, 1999, 40(3), 199 - 208
Articular cartilage proteoglycan metabolism in avian degenerative joint disease: effects of strain selection and body weight; Venkatesan N et al.; The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage . Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line) . Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis . By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks . Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater . Results with the feed-restricted group were intermediate between ad libitum-fed and J-line . Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT . The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.

J Cataract Refract Surg, 2000 Apr, 26(4), 613 - 5
Fungal keratitis after laser in situ keratomileusis; Sridhar MS et al.; A 22-year-old woman presented with pain, redness, watering, and decrease in vision in her left eye 15 days after laser in situ keratomileusis for myopia . Slitlamp examination showed a central full-thickness infiltrate with hyphate edges . Microscopic examination of corneal scrapings from the edge and underneath the flap showed fungal filaments, and the growth on culture media was identified as Scedosporium apiospermum.

J Vet Med Sci, 2000 Mar, 62(3), 281 - 5
Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses; Okumura M et al.; The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage . Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3) . Chondrocytes were centrifuged and cultured as pellets . Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks . The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/16H9 antibody, respectively . Concentration of GAG was significantly increased in the media of pellets stimulated by both IGF-Ialpha and IL-1alpha . Antigenic KS concentration was significantly increased in those stimulated by IL-1alpha, while no significant change was found in those stimulated by IGF-Ialpha . A high correlation between GAG and antigenic KS concentrations was found in the media of pellets stimulated by IL-1alpha (r=0.87), but not in those stimulated by IGF-Ialpha (r=0.43) . The results suggest that the concentration of antigenic KS reacting to 1/14/16H9 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage . The concentration of antigenic KS reacting to 1/14/16H9 antibody in biological fluids could therefore be a useful marker to further understand principally the catabolic and slightly the anabolic process of articular cartilage metabolism.

Hypertens Res, 2000 Mar, 23(2), 91 - 9
Tyrosine-kinase dependent TGF-beta and extracellular matrix expression by mechanical stretch in vascular smooth muscle cells; Joki N et al.; Vascular hypertrophy, which is characterized by proliferation of vascular smooth muscle cells (VSMC) and accumulation of extracellular matrix (ECM), is a major pathological change in blood vessels after chronic exposure to hypertension . Blood pressure is transmitted to the arterial walls and counterbalanced by mechanical stress, leading to stretching of circumferentially oriented VSMC, which may play some role in the pathogenesis of vascular hypertrophy . The present study was designed, therefore, to investigate the effect of mechanical stretch on the expression of ECM components and transforming growth factor-beta (TGF-beta), a potent stimulator for ECM production, and to examine the signal transduction mechanisms of the induction of TGF-beta in cultured rat VSMC . VSMC were subjected to cyclic stretch to provide a maximal elongation of 20% at a rate of 60 cycles per minute for up to 24 h . Mechanical stretch stimulated TGF-beta1 mRNA expression in a time- and elongation-dependent manner . Indeed, the secretion of TGF-beta proteins into the culture media was increased after stretch . Stretch also stimulated mRNA expression of the ECM components, type I and type IV collagen, and fibronectin, which was largely inhibited by addition of neutralizing antibody against TGF-beta . The tyrosine kinase inhibitors genistein and herbimycin A blocked the induction of TGF-beta1 and type I collagen by stretch, while protein kinase C inhibitors, the calcium channel blockers nitrendipine and gadolinium, or Ca removal from the media had no effect . These results suggest that stretch-induced, tyrosine kinase-mediated autocrine/paracrine production of TGF-8 may play a critical role in the progression of vascular remodeling associated with high blood pressure.

Exp Clin Endocrinol Diabetes, 2000, 108(1), 44 - 8
Effects of testosterone, FSH, and LH on oestradiol and progesterone secretion by preovulatory cumulus oophorus complexes of the rat; Regucka J et al.; Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study . They were killed in succession on the day of pro-oestrus at 11.00, 18.00, and 22.00 h . From ovarian preovulatory follicles cumulus oophorus complexes (COCs) were isolated and subsequently cultured with or without testosterone (T), T plus FSH, or T plus LH . In control cultures COCs isolated at all investigated hours released similar amounts of oestradiol . T stimulated this basal secretion and the effect was usually enhanced in the presence of FSH or LH . In control cultures the amount of released progesterone was greatest when expanded COCs were isolated (22.00 h) . T present in culture media diminished the amount of secreted progesterone . However, when T was added with FSH or LH a distinct stimulatory effect was observed, except in cultures with T plus FSH set up at 22.00 h . Previously, gonadotrophins alone did not effect progesterone secretion . The results suggest that T can regulate steroid, and especially progesterone secretion by COCs . Until the preovulatory gonadotrophin surge T can inhibit luteinization of COCs, while afterwards, acting synergestically with gonadotrphins (especially with LH), T can stimulate progesterone production in the cumulus granulosa cells.

Virus Genes, 2000, 20(1), 57 - 63
Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain; Pur B et al.; The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides . DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR . These fragments were assembled into a full-length cDNA clone in the yeast-E . coli shuttle vector pRS424, using homologous recombination in yeast . RNA produced by in vitro transcription of this clone was infectious upon electroporation into LLCMK2 cells, as shown by cytopathic effects and detection of viral antigens by indirect immunofluorescence, and by propagation of the virus released into the culture media . Biological properties of the transcript-derived virus, such as the pattern of dengue-specific protein synthesis and growth rate in LLCMK2 or C6/36 cells, resembled those of the parent DEN1 WP virus.

Oncology, 2000 Apr, 58(3), 261 - 70
Secretion of extracellular matrix (fibronectin), growth factor (transforming growth factor beta) and protease (cathepsin D) by hepatoma cells; Ito H et al.; We investigated facilitation of invasion by growth factors and chemotactic factors in tumor cell lines, particularly hepatocellular carcinoma . Hepatoma cells (PLC/PRF/5 and Hep G2) showed strong chemotaxis toward their respective conditioned media while metastatic pancreatic cancer cells (SU.86.86) and colon cancer cells (LS 174T) did not migrate toward their respective conditioned media . Based on immunoblotting, PLC/PRF/5 cells secrete fibronectin (an extracellular matrix constituent), transforming growth factor-beta (TGFbeta; a growth factor), and cathepsin D (a protease) . Fibronectin induced a migratory response in PLC/PRF/5 cells, and anti-fibronectin antibody abolished the migratory response of these cells to their conditioned medium . Anti-integrin-beta(1) antibody also impeded migration of these cells toward conditioned medium . Polyclonal anti-TGFbeta antibody and protease inhibitors (alpha(2)-macroglobulin and leupeptin) added to culture media-modulated secretion of fibronectin by PLC/PRF/5 cells . Although exogenous TGFbeta suppressed SU.86.86 cells, it enhanced PLC/PRF/5 cell adhesion to substrate, increasing viable cell numbers . These actions indicate that hepatocellular carcinoma may possess a forceful autocrine mechanism enabling cells to survive and proliferate under cirrhotic conditions .

Vet Parasitol, 2000 Apr 28, 89(3), 199 - 208
Comparison of growth rates of Tritrichomonas foetus isolates from various geographic regions using three different culture media; Lun Z et al.; The growth rates of 16 isolates of Tritrichomonas foetus from three distinct geographic regions were investigated in modified Diamond's medium, liver infusion broth medium and a commercially available culture kit . While some differences in growth characteristics were detected for different isolates and in the three different media, all isolates grew . Trichomonads reached peak concentrations from an initial concentration of 10(4) trichomonads/ml on Days 2, 3 and 4 in modified Diamond's medium, on Days 2-6 (excluding CAPTF102) in the commercial culture kit and on Days 2-7 in liver infusion broth medium . Viable parasites were detectable for longer periods in liver infusion broth medium and the commercial culture kit than in Diamond's medium . Peak concentrations for isolates tended to be higher in modified Diamond's medium than in liver infusion broth medium or the commercial culture kit . Results show that these three media are suitable for the growth of all 16 T . foetus isolates from three continents and suggest that these media could be used effectively throughout the world.

J Am Coll Cardiol, 2000 Apr, 35(5), 1338 - 46
Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes; Tatsumi T et al.; OBJECTIVES: The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes . BACKGROUND: Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells . However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility . METHODS: Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent . RESULTS: Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner . The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate) . These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis . Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters . The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes . Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes . KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects . CONCLUSIONS: These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.

Connect Tissue Res, 1999, 40(4), 259 - 72
Mature full-thickness articular cartilage explants attached to bone are physiologically stable over long-term culture in serum-free media; Dumont J et al.; Mature tissue explants containing the entire depth of articular cartilage, calcified and uncalcified, attached to a thin layer of subchondral bone were isolated from bovine humeral heads of 1-2-year-old steers . These explants were placed in defined serum-free culture medium for a period of 3 weeks to investigate their biological and mechanical stability and thus to determine their potential utility in studies of cartilage physiology . Tissue mass remained constant over the culture period and no evident tissue swelling or distortion was observed . Chondrocytes were viable in all zones at the time of tissue isolation and throughout the culture period, with the exception of a thin layer of cells at the articular surface and the cut radial edge of the disks . Proteoglycan metabolism attained a steady state after 5 days of culture when the rate of loss of proteoglycan to culture media was compensated by new synthesis to maintain a stable proteoglycan content . Collagen metabolism was also stable with a constant content of type II collagen and a constant content of denatured collagen II throughout culture; the content of the C-propeptide of type II procollagen as a measure of procollagen synthesis, dropped slightly during the first week to attain a steady state after that time . Dynamic and equilibrium mechanical properties of these explant disks were also stable confirming maintenance of these tissue properties during long-term culture . In addition, the disk geometry of the system, with the cut surface in the bone parallel to the intact articular surface, is well-suited to study tissue regulation by mechanical load . Taken together, the stability of these indicators of tissue physiology indicates the maintenance in serum-free conditions of normal metabolism for organ cultures containing full-depth mature articular cartilage attached to bone.

J Bone Miner Res, 2000 Mar, 15(3), 534 - 40
Vascular endothelial growth factor is expressed in human fetal growth cartilage; Garcia-Ramirez M et al.; Angiogenesis is a crucial event in endochondral ossification . Chemoattractants and mitogens for endothelial cells (such as basic fibroblast growth factor {bFGF} and transforming growth factor beta {TGF-beta}), which act as local regulators of the process, are synthesized by chondrocytes under several stimuli and in relation to the differentiation stage of the cartilage . Vascular endothelial growth factor (VEGF) is a 44-kDa protein well known as a potent angiogenic molecule owing to its mitogenic and permeability-causing properties . In this work, VEGF was located by immunohistochemistry in growth plate cartilage of human fetuses (20-22 weeks old) and its expression was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR) . Primary culture of human fetal epiphyseal chondrocytes (HFEC) maintained VEGF expression at protein and messenger RNA (mRNA) levels and this expression was stimulated by cartilage-promoting growth factors incorporated into the culture media (rFGF-b, rTGF-beta1, and insulin-like growth factor {rFGF-b} at 50 ng/ml) . The conditioned medium (CM) of HFEC stimulated the proliferation of endothelial cells, and this was partially blocked by anti-VEGF antibody . These studies showed VEGF production by chondrocytes of the epiphyseal growth cartilage and suggested a role of this factor in cartilage physiology and the angiogenic process.

J Endocrinol, 2000 Apr, 165(1), 101 - 13
Expression of mRNA encoding IGF-I, IGF-II and type 1 IGF receptor in bovine ovarian follicles; Armstrong DG et al.; IGFs regulate gonadotrophin-stimulated proliferation and differentiation of granulosa and theca cells in vitro . However, the detailed pattern of mRNA expression of IGFs in bovine follicles remains controversial . The objectives of this study were therefore to describe the temporal and spatial pattern of expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor in bovine follicles in vivo . The expression of mRNA encoding IGF-II was detected in theca tissue from around the time of antrum formation up to and during the development of dominance . No IGF-II mRNA expression was detected in granulosa cells . In the majority of follicles we were unable to detect mRNA encoding IGF-I in either granulosa or theca tissue from follicles at any stage of development . Occasionally low amounts of mRNA encoding IGF-I were detected in the theca externa and connective tissue surrounding some follicles . Type 1 IGF receptor mRNA was detected in both granulosa and theca cells of preantral and antral follicles . Expression was greater in granulosa tissue compared with theca tissue . We also measured IGF-I and -II mRNA in total RNA isolated from cultured granulosa and theca cells using reverse transcriptase PCR . In contrast to the in vivo results, IGF-II mRNA was detected in both granulosa and theca tissue . IGF-I mRNA was detected in theca tissue and in very low amounts in granulosa cells . Using a specific IGF-I RIA we were unable to detect IGF-I immunoreactivity in granulosa conditioned cell culture media . Using immunohistochemistry we detected IGF-I immunoreactivity in some blood vessels within the ovarian stroma . We conclude from these results that IGF-II is the principal intrafollicular IGF ligand regulating the growth of bovine antral follicles . In preantral follicles the expression of mRNA encoding type 1 IGF receptor but absence of endogenous IGF-I or -II mRNA expression, highlights a probable endocrine mechanism for the IGF regulation of preantral follicle growth.

J Neuropathol Exp Neurol, 2000 Feb, 59(2), 170 - 4
Immunocytochemical, ultrastructural and neurochemical evidences on synaptogenesis and dopamine release of rat chromaffin cells co-cultured with striatal neurons; Zhang L et al.; The results reported herein address the question of synaptogenesis between adrenal chromaffin cells and striatal neurons . The release of dopamine from chromaffin cells in the presence of striatal neurons was also examined . Co-culture of newborn rat chromaffin cells and striatal neurons at 1:1 ratio was made . Cultures were examined morphologically using immunocytochemistry and ultrastructural techniques (transmission electron microscopy), while quantitation of dopamine in the culture media by HPLC-ECD was also determined . Neurite outgrowth from chromaffin cells was enhanced in the presence of striatal neurons and numerous synaptic-like contacts between these two cell types were observed . Higher concentration of dopamine was also present in the co-culture medium as compared with those containing only chromaffin cells . The development of synapses between these two types of cells may give support to the functionality of transplants in human cases of Parkinson disease (PD).

Equine Vet J, 2000 Mar, 32(2), 140 - 50
Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints; von Rechenberg B et al.; Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance . Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity . Slight discolouration and fibrillation or minor clefts of the cartilage were considered as moderate OA, whereas erosions of articular cartilage down to the subchondral bone were considered as cases of severe OA . Explant cultures of equine synovial membrane and articular cartilage released the local mediators, NO and PGE2, as well as detectable levels of NMP activity into culture media . Concentrations of NO were higher in articular cartilage explants compared to synovial membrane explants, whereas concentrations of PGE2 were higher in synovial membrane explants . The NMPs with collagenolytic activities were similar in both explant cultures, whereas gelatinolytic activities were higher in synovial membrane explant cultures and caseinolytic activities were generally higher in articular cartilage explant cultures . Furthermore it was shown that concentrations or enzyme activities increased according to the severity of disease of the joints . Concentrations for NO, collagenolytic and gelatinolytic NMPs were relatively stable, whereas PGE2 and caseinolytic NMP concentrations increased over time in culture.

Hum Reprod, 2000 Apr, 15(4), 881 - 9
Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro; Tao T et al.; The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst . Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) . The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated . Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres . No major differences were found between the three basic culture media . In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively) . Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3 . 7 versus 57.8 +/- 3.3 total cells) . The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%) . In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes . These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.

Hum Reprod, 2000 Apr, 15(4), 846 - 52
Laser-induced immobilization and plasma membrane permeabilization in human spermatozoa; Montag M et al.; We evaluated the potential use of a non-contact, 1.48 microm wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media . When we applied a single laser shot near to the middle region of the sperm tail, spermatozoa could be immobilized either temporarily or permanently, depending on the energy used . Above an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane . We then explored the use of a double laser shot technique . Spermatozoa were temporarily immobilized by a first laser shot applied near to the sperm tail followed by permeabilization with a second laser shot aimed directly at the sperm tail . This sequential approach yielded permanent immobilization at much lower energy values compared with the single shot technique . Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation . We conclude that a non-contact 1.48 microm diode laser system can be used for immobilization of spermatozoa and for permeabilization of the sperm tail membrane . This laser procedure may offer an alternative to currently used sperm pretreatment prior to intracytoplasmic sperm injection.

J Cell Physiol, 2000 May, 183(2), 172 - 81
Expression and release of insulin-like growth factor binding proteins in isolated epiphyseal growth plate chondrocytes from the ovine fetus; De Los Rios P et al.; Insulin-like growth factor-II (IGF-II) is an autocrine modulator of epiphyseal chondrogenesis in the fetus . The cellular availability of IGFs are influenced by the IGF-binding proteins (IGFBPs) . In this study, we investigated the control of expression and release of IGFBPs from isolated epiphyseal growth plate chondrocytes from the ovine fetus by hormones and growth factors implicated in the chondrogenic process . Chondrocytes were isolated from the proliferative zone of the fetal ovine proximal tibial growth plate and maintained in monolayer culture at early passage number . Culture media conditioned by chondrocytes under basal conditions released IGFBPs of 24, 34, and 29 kDa, and a less abundant species of 39-43 kDa that were identified immunologically as IGFBP-4, IGFBP-2, IGFBP-5, and IGFBP-3, respectively . Messenger RNAs encoding each species were identified by Northern blot analysis within chondrocytes, as was mRNA encoding IGFBP-6 . Exposure to IGF-I or IGF-II (13 or 26 nM) caused an increase in expression and release of IGFBP-3 . The release of IGFBP-2 and IGFBP-5 were also potentiated without changes to steady state mRNA, and for IGFBP-5 this was due in part to a release from the cell membrane in the presence of IGF-II . Insulin (16.7 or 167 nM) selectively increased mRNA and the release of IGFBP-3, while cortisol (1 or 5 microM) inhibited both mRNA and release of IGFBP-2 and IGFBP-5 . Transforming growth factor-beta1 (TGF-beta1) (0.1 or 0.2 nM) increased the expression and release of IGFBP-3, and caused an increase in mRNAs encoding IGFBP-2 and IGFBP-5 . Neither growth hormone (GH), fibroblast growth factor-2, nor thyroxine (T(4)) had any effect on IGFBP expression or release . The results suggest that IGFBP expression and release within the developing growth plate can be modulated by IGF-II and other trophic factors, thus controlling IGF availability and action .

Immunopharmacol Immunotoxicol, 2000 Feb, 22(1), 103 - 15
Immunomodulatory effect of the homoeopathic drug Engystol-N on some activities of isolated human leukocytes and in whole blood; Fimiani V et al.; Engystol-N at the doses of 10(-4) and 10(-8) in isolated human leukocytes stimulates the superoxide anion generation by neutrophils and the cytokine(s) production by T lymphocytes . In whole blood the same concentrations of the drug produce the decrease of the superoxide anion generation of neutrophils, this inhibiting activity appears 6 h after the administration of the drug and persists only in presence of lymphocytes . Culture media of T lymphocytes treated with Engystol-N show the same inhibiting effect on superoxide anion generation by neutrophils . From these data it is possible to conclude that the drug stimulates the secretion of lymphokine(s) with inhibiting action on superoxide anion generation of neutrophils that prevail over the direct stimulating effect, confirming and extending the immunomodulatory ability of the drug.

Theriogenology, 1999 Oct 1, 52(5), 847 - 61
Effect of culture media on embryo development from prepubertal goat IVM-IVF oocytes; Izquierdo D et al.; Experiments were carried out to develop and improve in vitro culture systems for IVM-IVF prepubertal goat oocytes . Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats . Oocytes were matured in TCM-199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air . Matured oocytes were placed in drops of TALP- fert medium supplemented with hypotaurine (1 microgram/mL) and inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al . (69) but with 100 micrograms/mL heparin . At 24 h post insemination the ova were transferred to various in vitro culture media, and early embryo development was evaluated until Day 8 post insemination . Specifically, in the studies described here, we have compared the effects of (Experiment 1) co-culture systems using oviductal ephitelial cells (OEC) and cumulus cells (CC), both caprine and bovine; (Experiment 2) the presence of serum and/or OEC; (Experiment 3) 4 culture media (TCM199, Ham's F10, CZB abd SOF) for co-culture with OEC; and (Experiment 4) conditioned medium with OEC . In Experiment 1, the percentage of morulae plus blastocysts was higher in culture with OEC, both caprine and bovine (15.1 and 14.8%, respectively) than with CC (4.1 and 6.7%, respectively) . In Experiment 2, the OEC with EGS did not improve the percentage of morulae and blastocysts obtained with OEC alone (14.3 and 23.1% respectively) . In Experiment 3, this percentage was higher using OEC with TCM-199 compared to CZB medium (21.3 and 12.3%, respectively) and in Experiment 4, the results were 3.7, 11.2 and 21.3% for TCM-199 without cells, Conditioned Medium and co-culture with OEC, respectively.

Theriogenology, 2000 Jan 15, 53(2), 619 - 26
Interactions between embryos and the culture milieu; Bavister BD; Although in vitro production of embryos up to the blastocyst stage is now possible in numerous species, the quality and quantity of embryos are still not satisfactory . Clearly, culture conditions do not yet replace all of the benefits of development within the female reproductive tract . Analysis of the interactions between embryos and the components of culture media provides insights into regulatory mechanisms and how they are perturbed in vitro, and also offers some clues about the nature of the support provided to early embryos by the female tract . Further elucidation of these events and their underlying regulation will be helpful for improving culture media formulations to support normal embryo development in vitro.

Theriogenology, 2000 Jan 15, 53(2), 575 - 97
Effects of different reproduction techniques: AI MOET or IVP, on health and welfare of bovine offspring; van Wagtendonk-de Leeuw AM et al.; Since the introduction of in vitro production (IVP) of bovine and sheep pre-implantation embryos, increased birth weights and other deviations of IVP calves and lambs compared with AI or MOET offspring have been reported . Study 1 of the present paper, a comparison between AI, MOET and IVP (co-culture/serum) calves with respect to calf and calving characteristics in large-scale field conditions, confirms these reports . In addition, it is shown that MOET calves tend towards higher birth weights and have significantly longer gestations and more difficult calvings than AI calves . It is presently unknown if the effect of IVP is also observed later in life . In this paper, data on reproduction characteristics of bovine IVP co-culture/serum offspring are presented . Semen production--and non return data of one year old IVP bulls and superovulation-, AI- and OPU/IVP results of one year old IVP heifers are compared with those of one year old AI and MOET animals producing semen or embryos in the same time period . So far, there are no indications that the use of IVP is reflected in deviate reproduction characteristics of bovine IVP offspring . It has been suggested that use of co-culture cells and serum during in vitro culture of bovine (and sheep) embryos may partially explain the increased birth weights and other deviations of bovine and sheep IVP offspring . Deletion of these factors in semi-defined culture media, e.g . Synthetic Oviductal Fluid (SOF), could result in more normal offspring . Study 2 investigates this hypothesis in both field conditions (Study 2a, comparing AI, IVP co-culture/serum and IVP SOF calves) and in semi-standardized conditions (Study 2b, comparing MOET, IVP co-culture/serum and IVP SOF calves at one herd) . In Study 2a, although IVP SOF calves showed (non-significant) shorter gestations, easier calvings and lower percentages of perinatal mortality and congenital malformations than IVP co-culture calves, birth weights were not decreased . In Study 2b however, the difference between IVP co-culture and IVP SOF calves in birth weight and ease of calving was significant (P < 0.05), IVP SOF calves resembling MOET calves more . IVP calves differed significantly from MOET calves with respect to several physiological parameters, such as blood oxygen saturation level, heart beat frequency and some measures of the heart . In addition, in Study 2b, recipients receiving an IVP SOF embryo showed a more regular return to estrus than those receiving an IVP co-culture embryo . From Study 2 it can be concluded that using a semi-defined medium for in vitro culture (SOF) may improve characteristics of IVP calves born.

Theriogenology, 1998 Jul 15, 50(2), 293 - 300
Bacteria in semen used for IVF affect embryo viability but can be removed by stripping cumulus cells by vortexing; Kim IH et al.; Bacterial contamination of in vitro vs in vivo produced embryos presents a particular danger because of the alteration of the zona pellucida and the use of various biological products during culture . Our objective was to investigate the effects of semen contaminated with bacteria on IVF of bovine oocytes and to determine if removal of cumulus cells by vortexing as opposed to pipetting would reduce contamination and improve subsequent embryonic development . Semen from 5 bulls of the Native Korean breed (Bulls A, B, C, D, E) was used for IVF of matured oocytes . Preliminary studies had shown that the semen from Bulls A, B, D and E but not Bull C was contaminated with various species of common bacteria . After IVF, the cumulus cells surrounding the oocytes were removed either by pipetting or vortexing . Viability and cleavage rates of the resulting zygotes was assessed after 44 h in culture . When cumulus cells were removed by pipetting, only zygotes derived from oocytes that were fertilized with uncontaminated semen from Bull C developed to morula and blastocyst stages; zygotes derived from oocytes that were fertilized with contaminated semen from Bulls A, B, D and E started to degenerate, and the culture media became noticeably turbid . When cumulus cells were removed by vortexing, zygotes derived from oocytes fertilized with either contaminated or uncontaminated semen showed good rates of development (16 to 32%) to morula or blastocyst stages . From these results it can be concluded that the bacteria introduced with the semen contaminated the in vitro system and severely reduced the viability of the embryos . In contrast, complete removal of the cumulus cells with vortexing, as opposed to pipetting, reduced the contamination of the culture medium, allowing embryonic development to take place.

Theriogenology, 1998 Jul 15, 50(2), 213 - 22
The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes; Katska L et al.; The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles . Methods of culturing preantral follicles are now being developed . The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth . Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns . The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2) . The total number of isolated follicles of different size classes was similar in heifers and cows . No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2) . However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium . The survival time of follicles was related to their initial size at the beginning of culture . The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS) . Survival and growth of some follicles was maintained for 23 d.

Life Sci, 2000 Feb 11, 66(12), 1127 - 37
Quantitative analysis of matrix metalloproteinases-2 and -9, and their tissue inhibitors-1 and -2 in human placenta throughout gestation; Niu R et al.; To elucidate the implication of type IV collagenases(MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) for placental development, we quantified their levels in the conditioned media of placental organ culture and primary culture of the trophoblast as well as in the tissue extracts of placentas from different stages of gestation using specific enzyme-linked immunosorbent assays . First trimester villous tissue secreted about 10 times more pro-MMP-2 than pro-MMP-9, and pro-MMP-2 levels dramatically decreased in the second trimester . On the other hand, pro-MMP-9 levels were more than 10 times higher than those of pro-MMP-2 in the primary culture of the first trimester trophoblast, indicating the involvement of stromal cells for prominent pro-MMP-2 secretion from first trimester villous tissue described above . Levels of TIMPs, especially those of TIMP-2, remained constant throughout gestation both in the culture media and tissue extracts . Gelatin zymography revealed abundant secretion of the active form of MMP-2 as well as pro-MMP-2 from first trimester villous tissue . Western immunoblot analysis confirmed the presence of both TIMP-1 and TIMP-2 in placental tissue . These results suggest that active secretion of MMP-2 from villous tissue in the first trimester and constant production of TIMPs throughout gestation are characteristic of placental development.

Theriogenology, 1998 Jun, 49(8), 1525 - 36
Production of sheep embryos in vitro and development of progeny following single and twin embryo transfers; Brown BW et al.; An in vitro culture system for producing ovine embryos is described, in which heat inactivated sheep serum was used as a protein source for maturation, fertilization and 7-d culture phases . Ovaries obtained from a commercial abattoir were used as the source of mature ewe (285) and lamb oocytes (356), which were subsequently cultured in this system to yield similar mean cleavage rates of 91 and 92%, respectively, but significantly different (P < 0.025) proportions for blastocysts/cleaved oocytes (46 and 18%) . At Days 7 or 8 of culture, embryos from each source were transferred, either singly (ewe-derived) or in pairs (ewe- and lamb-derived), to hormonally synchronized recipient ewes, resulting in the birth of lambs ranging in weight from 2.5 to 8.8 kg for singletons and 2.6 to 8.0 kg for twins . Mean gestation length of 153.4 +/- 0.5 d (range 151 to 160) was slightly longer than the expected norm of about 150 d . The pregnancy rate was significantly higher after the transfer of embryo pairs (64%) than single (39%) embryos, while survival of lambs to weaning was greater for singleton (80%) than for twin lambs (43%) . Some factor(s) in the culture media, such as growth factors in the sera, may have a mitogenic effect on embryonic cells, or it may alter the distribution of cells to the trophectoderm and inner cell mass, resulting in enhanced body growth rates.

Theriogenology, 1998 Jun, 49(8), 1489 - 99
Effect of hyaluronic acid on development of in vitro produced bovine embryos; Furnus CC et al.; The present study was carried out to evaluate the effect of hyaluronic acid (HA) added to the culture medium on bovine embryo development to the blastocyst stage as well as embryo quality and viability after freezing and thawing . In vitro matured and fertilized (IVM/IVF) bovine oocytes from slaughterhouse ovaries were cultured for 8 d in SOFm supplemented with 4 mg/mL fatty acid-free BSA, either in the absence or presence of 1 or 0.5 mg/mL HA . There was a significant increase in blastocyst yield in the presence of 1 mg/mL HA (P < 0.01), whereas 0.5 mg/mL HA was ineffective . Cleavage rate and mean number of days to blastocyst formation were unaffected by HA at any concentration . At 1 mg/mL, HA did not affect either post-freeze survival of Grade 1 and 2 blastocysts or the number of nuclei per blastocyst . Supplementation with HA at 1 mg/mL also significantly enhanced embryo development up to the blastocyst stage (P < 0.05) in a chemically-defined culture medium without a protein source . It is concluded that supplementation of both semi-defined and defined culture media with 1 mg/mL HA improves the development of IVM/IVF bovine embryos to the blastocyst stage, without affecting embryo quality and post-freeze survival . These results open the possibility of including HA in culture media in order to increase the efficiency of in vitro blastocyst production from in vitro-matured bovine oocytes.

Mech Ageing Dev, 2000 Feb 22, 114(1), 37 - 48
Endothelin-1 in monolayer cultures of articular chondrocytes from young and old rats: regulation by growth factors and cytokines; Messai H et al.; The endothelin-1 (ET-1) concentrations were measured by RIA in the media of confluent monolayer cultures of rat articular chondrocyte (RAC) exposed to fetal calf serum (FCS) and several growth factors and cytokines . The cells were obtained from 1- and 18-month-old rats . First passage cells were starved in Dulbecco's modified Eagle's medium (DMEM) containing 0.2% FCS serum for 24 h and then incubated for 48 h in the same fresh medium with each of the following factors: fetal calf serum (FCS), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and NO donor, sodium nitroprusside (SNP) . The following was found: the cells from 18-month-old animals accumulated about twice as much ET-1 per microg DNA under basal (low serum) and stimulated conditions as cells from young rats . All, but PDGF and SNP produced concentration-dependent rise in ET-1 levels, the most effective being 10% FCS, IL-1beta, TNF-alpha, EGF, IGF-1 and LPS . TGF-beta caused the smallest stimulation and PDGF was ineffective or slightly inhibitory at high concentrations . SNP caused concentration-dependent decrease of ET-1 concentrations . ET-1-specific mRNA was identified by RT-PCR in cells incubated with the above factors and its concentration paralleled that of the peptide . This suggests that ET-1 found in the culture media of RAC stems, at least in part, from the synthesis . Increased immunoreactive peptide concentration and mRNA expression with the age of the donor rat and its regulation by several growth factors and cytokines suggest the involvement of ET-1 in chondrocytes' physiology and possibly pathology.

J Am Coll Cardiol, 2000 Mar 15, 35(4), 968 - 73
Expression of vascular endothelial growth factor in patients with acute myocardial infarction; Hojo Y et al.; OBJECTIVE: The purpose of this study was to investigate the clinical significance of vascular endothelial growth factor (VEGF) in acute myocardial infarction (AMI) . We also examined the involvement of peripheral blood mononuclear cells (PBMCs), which are a possible source of VEGF in AMI . BACKGROUND: VEGF is a potent endothelial cell-specific mitogen and could affect the outcome of AMI . METHODS: Thirty patients with AMI were used for this study . Serum and PBMCs were isolated from peripheral blood on days 1, 7, 14 and 21 after the onset of AMI . PBMCs were cultured at a density of 5 x 10(6) cells/ml for 24 h . VEGF levels in serum and the culture media were measured by enzyme-linked immunosorbent assay using a specific anti-human VEGF antibody . RESULTS: Serum VEGF levels elevated gradually after the onset of AMI and reached a peak on day 14 . VEGF levels in the culture medium of PBMCs after incubation for 24 h (PBMC-VEGF) were maximally elevated 7 days after the onset . Maximum serum VEGF levels showed significant positive correlations with maximum creatine phosphokinase (CPK) levels (r = +0.70, p < 0.001), but maximum PBMC-VEGF levels did not correlate with maximum CPK levels . Patients showing improvement in left ventricular systolic function during the course of AMI showed significantly higher PBMC-VEGF levels than patients without improvement . CONCLUSIONS: The extent of myocardial damage contributes to the elevation of serum VEGF levels in AMI . VEGF produced by PBMCs may play an important role in the improvement of left ventricular function by promoting angiogenesis and reendothelialization after AMI.

Theriogenology, 1998 Sep, 50(4), 659 - 66
Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro; Chrenek P et al.; Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated . Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females . The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova) . However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1) . The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2) . In serum-free, growth factor-supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%) . In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture . Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively) . Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.

Theriogenology, 2000 Mar 1, 53(4), 877 - 85
Placental lactogen as a regulator of luteal cells function during pregnancy in sheep; Gregoraszczuk EL et al.; The luteotropic activity of ovine placental lactogen (oPL) on different days of gestation in ewes was assessed using in vitro methods . Corpora lutea (CL) harvested on Days 45, 70, 95, 120 and 135 of gestation and during parturition were enzymatically dispersed and plated on multiwell plates . After 48 h of incubation, all cultures were terminated and media were frozen for further steroid analysis . Cells were cultured in control medium, with addition of oPL alone, or in combination with PGE2 or PGF2alpha . Supplementation of culture media with oPL increased basal progesterone secretion by cells isolated on Days 45 and 70 of gestation . There was no effect on progesterone secretion by cells isolated on other days of gestation; PGE2 added to the culture media increased progesterone production only by cells isolated on Day 70 of pregnancy . Simultaneous oPL treatment with PGE2 had a statistically significant and stimulatory effect on progesterone production by luteal cells collected on Days 70 and 95 of pregnancy . In contrast, PGF2alpha alone in culture media decreased progesterone secretion by cells isolated on Days 45, 70 and 95 of gestation, while oPL plus PGF2alpha on Days 70 and 95 of gestation protected against luteolytic action of PGF2alpha . The results showed 1) a direct effect of the oPL on luteal cells isolated on Days 45 and 70 of gestation; 2) synergism between PL and PGE2 in progesterone production; by cells isolated on Day 70; 3) and a luteoprotective effect of oPL against the luteolytic action of prostaglandin F (PGF2alpha) observed on Days 70 and 95 of gestation.

Theriogenology, 1999 May, 51(7), 1375 - 90
In vitro production of pig embryos: comparisons of culture media and boars; Long CR et al.; The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology . Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects . To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data . Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively . Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2% . Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts . For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS . These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively . These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro . Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent . Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.

Theriogenology, 1999 May, 51(7), 1363 - 74
Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro; Kuzmina TI et al.; At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals . We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ({Ca2+}is) in the oocytes . Cumulus-oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3) . Levels of {Ca2+}is in oocytes were determined by using the fluorophore chlortetracycline . The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3) . Compared with the control, lower levels of {Ca2+}is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs . 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs . 1.52+/-0.04 AU in System 3, P<0.001) . These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in {Ca2+ }is levels at early stages of oocyte maturation.

Theriogenology, 1999 Apr 1, 51(5), 911 - 26
Development and application of competitive ELISA assays for rat LH and FSH; Pappa A et al.; Rat LH (rLH) and FSH (rFSH) were measured by sensitive and specific competition ELISAs . The rat LH ELISA used rLH-I-9 coated plates, an antiserum against rLH and an antibody against rabbit IgG labeled with peroxidase . Using rLH-RP-3 as a standard, rat LH was determined by binding of the anti-LH antibody to rLH-I-9 coated plates . The sensitivity of the assay was 0.8 ng/mL . Similarly, the rat FSH-ELISA used rFSH-I-8 coated plates, an antiserum against rFSH and an antibody against rabbit IgG labeled with peroxidase . Using rFSH-RP-3 as a standard, the FSH-ELISA was also determined by binding of the anti-FSH antibody to rFSH-I-8 coated plates . The sensitivity of this assay was 1.25 ng/mL . Both rat LH and FSH ELISA assays are highly specific and provide accurate determination of gonadotrophins in buffers, sera, cell culture media, and anterior pituitary extracts . These assays were used for monitoring the gonadotrophin surge-attenuating factor (GnSAF) and inhibin activities present in human follicular fluid (hFF) . The 2 new ELISA procedures have practical advantages (safety, convenience, economy) over the RIA methods, and they perform as well as the RIA techniques at the same range of concentrations.

Arthritis Rheum, 2000 Mar, 43(3), 673 - 82
Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1); Dahlberg L et al.; OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 {MMP-1}) . METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis . The articular cartilage samples were cultured in serum-free medium . A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media . In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1 . Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage . RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples . In contrast, proteoglycan (GAG) release was not increased . This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples . The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples . Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage . Proteoglycan loss was unaffected by either inhibitor . Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity . CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1 . The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules . Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors . Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.

Arthritis Rheum, 2000 Mar, 43(3), 664 - 72
Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase; Billinghurst RC et al.; OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes . METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 {MMP-8} and MMP-13, respectively) but not of collagenase 1 (MMP-1) . Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media . A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG) . RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage . A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha . In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation . The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage . CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss . It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage . Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis . Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.

Horm Res, 1999, 52(4), 178 - 85
Secretion of noncomplexed 'Big' (10-18 kD) forms of insulin-like growth factor-II by 12 soft tissue sarcoma cell lines; Elmlinger MW et al.; The paraneoplastic production of pro-insulin-like growth factor-II (IGF-II) forms causes tumour hypoglycaemias and presumably also has an effect on tumour cell growth . We investigated the molecular weights of IGF-II forms and their ability to form complexes with IGF binding proteins (IGFBPs) in conditioned culture media (CM) from 12 paediatric soft tissue sarcoma (STS) cell lines and from two healthy fibroblast lines . Untreated CM were separated by size exclusion chromatography using biocompatible HPLC . Subsequently, IGF-II, IGFBP-2 and IGFBP-3 were determined in the HPLC fractions by specific RIAs . In the CM, IGF-II concentrations between 0.5 and 8.6 ng/10(6) cells were measured but no IGF-I was detectable . Parallel to this investigation, a high IGF-II mRNA level averaging 44.4 +/- 29.7% was measured by semi-quantitative RT-PCR . The STS cell lines secreted a higher proportion of big-IGF-II forms reaching 10-18 kD (10-33% of the total IGF-II secreted) compared to the healthy fibroblasts (2.5-5%) . At the same time, the proportion of IGF-II bound with IGFBP in complexes of 35- 70 kD and 150 kD was reduced by up to 85% in CM from tumour cells . The tumour cell lines apparently secrete a different spectrum of IGF-II forms than healthy fibroblasts . The reduced ability to form complexes with IGFBP and the higher molecular weight of the IGF-II forms produced by the tumour cells indicate that these forms could in fact be the known tumour-associated pro-IGF-II forms . Due to these characteristics, the big-IGF-II forms probably have an altered biological effect on the tumour cells when compared to IGF-II .

J Agric Food Chem, 2000 Mar, 48(3), 951 - 7
Heterologous expression in Aspergillus nidulans of a Trichoderma longibrachiatum endoglucanase of enological relevance; Villanueva A et al.; An Aspergillus nidulans transformant expressing the Trichoderma longibrachiatum endoglucanase 1 gene (egl1) has been constructed . The extracellular production of EGL1 in different culture media has been studied, and a medium has been found in which EGL1 is the predominant extracellular protein produced . The enzymatic properties of the heterologously produced EGL1 are very similar to those of the native enzyme . Grape maceration in the presence of culture filtrate enriched in EGL1 resulted in increased release of aroma precursors, particularly in the case of aromatic grapes . Cryoscanning electron microscopy of the flesh of grapes treated with EGL1-enriched culture filtrate revealed degradation of the cell wall matrix.

Osaka City Med J, 1999 Jun, 45(1), 15 - 23
Effect of saliva on the growth of Helicobacter pylori; Morii H et al.; The effect of saliva mixed in culture media from young healthy volunteers on the growth of Helicobacter pylori (H . pylori) was investigated . Saliva was centrifuged at 15,000 rpm for 10 min and the supernatant was mixed with the reference strain of standard H . pylori which was suspended in sterilized physiological saline . The mixture was inoculated onto agar plate and incubated for 3 days . The numbers of colonies were counted . Saliva from a young male volunteer stimulated the growth of H . pylori significantly compared with the control . This result indicates that there may be such individuals who have saliva increasing the growth of H . pylori . But this is the preliminary report and more studies are needed to know what factors in saliva may affect the growth of H . pylori.

Brain Res Mol Brain Res, 2000 Mar 10, 76(1), 180 - 90
Constraints on proper folding of the amino terminal domains of group III metabotropic glutamate receptors; Peltekova V et al.; The glutamate binding site of the G-protein coupled metabotropic glutamate receptors (mGluRs) is contained within the large extracellular amino terminal domain (ATD) of the receptor . In this study, we examined the ligand binding properties and cellular dispositions of the membrane-bound mGluR4 and mGluR8 subtypes of mGluRs, and a series of truncated versions of these receptors . Truncation of the ATDs of mGluR4 and mGluR8 40 amino acids upstream of the first transmembrane domain produced soluble proteins that were secreted into the cell culture media of transfected human embryonic kidney cells . The soluble receptors retained ligand binding capabilities . Additional constructs of the ATDs of mGluR4 and mGluR8 were assessed for their ability to bind the agonist {(3)H}L-AP4 and for secretion from cells . A shorter mGluR4 construct truncated 98 amino acids upstream from the first transmembrane domain failed to bind {(3)H}L-AP4, while the analogous mGluR8 construct displayed a low level of binding . Unlike the full-length receptors, which were expressed on the cell surface, or the soluble constructs which were secreted, the shorter constructs were primarily associated with intracellular membranes . These observations suggest that the cysteine-rich region may be important for efficient secretion, but not absolutely obligatory for ligand binding . Surprisingly, longer constructs encoding the entire ATDs of mGluR4 and mGluR8 failed to bind ligand and were localized intracellularly . Together, these findings demonstrate that there are strict limitations on the proper folding of truncated versions of the ATDs of mGluR4 and mGluR8 . Specifically, all of the leucine-isoleucine-valine binding protein homology region, and part of the cysteine-rich region is required for optimal secretion in a soluble form that retains ligand binding activity.

Thromb Res, 2000 Apr 15, 98(2), 203 - 11
Expression and purification of recombinant rabbit factor VII; Ruiz SM et al.; To facilitate studies of the in vivo role of the extrinsic pathway of coagulation in experimental hemostasis and thrombosis, a full-length cDNA-encoding rabbit factor VII was isolated using polymerase chain reaction-mediated DNA amplification from plaque-purified lambda gt11 phage . Repeated DNA sequencing of both full-length rabbit factor VII cDNA and shorter cDNA fragments verified four changes in the previously reported amino acid sequence of mature rabbit factor VII, now predicted to be 405 amino acids in length . Rabbit factor VII cDNA was transfected into human embryonic kidney 293 cells and a cell line that permanently expressed rabbit recombinant factor VII was established . Rabbit recombinant factor VII was purified from tissue culture media using a combination of barium citrate precipitation, DEAE-sepharose FF chromatography, benzamidine agarose, and affinity chromatography using a sheep antirabbit factor VII polyclonal antibody . The purity and authenticity of rabbit recombinant factor VII was confirmed by polyacrylamide gel electrophoresis and Western blot analysis . Homogeneous rabbit recombinant factor VII was fully active biologically as determined by prothrombin time assay in factor FVII-depleted plasmas, of both human and rabbit origin, using either human or rabbit thromboplastin . Rabbit recombinant factor VII should prove useful for future in vivo investigations of experimental coagulopathies.

J, Exp . Mar . Biol . Ecol. . 2000 Apr 5, 246(2), 145 - 161
Copper and iron concentrations in Ascophyllum nodosum (Fucales, Phaeophyta) from different sites in Ireland and after culture experiments in relation to thallus age and epiphytism; Stengel DB et al.; In laboratory experiments, copper concentrations in plants of Ascophyllum nodosum (L.) Le Jolis (Fucales, Phaeophyta) increased with the concentrations in the culture media and were highest in younger, meristematic thallus parts . After initial accumulation in high-copper medium and subsequent transfer to clean seawater for 5 days, no release of copper could be detected . Iron concentrations in A . nodosum tissue were not related to concentrations in the culture medium . Differences between copper concentrations in plants from different sites in areas with high yachting activity in Strangford Lough, Northern Ireland, could be explained by differences in water motion and human activity, in particular the application of copper-releasing antifouling paints to leisure boats . Iron concentrations were also highest in plants from the sheltered, polluted site but did not differ significantly between the other two sites . No differences in copper nor iron concentrations were found between different-aged thallus parts of plants from any site . X-ray microanalysis revealed that most of the iron detected was located in epiphytic pennate diatoms on the A . nodosum surface . In thallus areas without diatoms, iron levels were below the detection limit for X-ray microanalysis . Mapping for copper indicated that most of the accumulated copper was located in cells near and immediately below the thallus surface . "Epidermis"-shedding occurred in plants from the culture experiments and also in freshly-collected material and may have resulted in a loss of metal ions accumulated by surface cells and by epiphytic diatoms . The results suggest that A . nodosum could be used as a biological indicator for copper but not for iron, and that young, apical plant parts are most sensitive to changes in metal concentrations in the water.

Mol Biol Cell, 2000 Mar, 11(3), 819 - 31
Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state; Altschuler Y et al.; MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells . In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments . To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc . Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans . Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1 . However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected . Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis . MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections . Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed . These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.

Obstet Gynecol, 2000 Mar, 95(3), 353 - 7
Amniotic fluid--soluble vascular endothelial growth factor receptor-1 in preeclampsia; Vuorela P et al.; OBJECTIVE: To measure the levels of the soluble receptor for the potent angiogenic agent vascular endothelial growth factor (VEGF) in amniotic fluid (AF) in healthy and complicated pregnancies, and compare them with levels of erythropoietin, another factor upregulated by hypoxia . METHODS: We assessed amniotic fluid from the second (n = 35, gestational weeks 14-19) and third (n = 29) trimesters of healthy women, and from the third trimesters of preeclamptic (n = 22) and diabetic women with (n = 11) or without preeclampsia (n = 34) and from women with fetal growth restriction (FGR) (n = 14) for soluble VEGF receptor-1 (VEGFR-1) by enzyme-linked immunosorbent assay . RESULTS: In early normal pregnancy, AF-soluble VEGFR-1 levels were higher (median 22 ng/mL, range 2.3-29.5 ng/mL) than in the third trimester (median 13 ng/mL, range 0.5-32 ng/mL; P < .05) . In preeclamptic women during the third trimester, levels were higher (median 20 ng/mL, range 10.5-37 ng/mL; P < .05) than healthy controls . The lowest third-trimester levels were in diabetic women (median 11 ng/mL, range 0.5-27 ng/mL) . In women with preeclampsia and diabetes, AF-soluble VEGFR-1 levels remained lower (median 13, range 6-32 ng/mL; P < .05) than in women with preeclampsia alone . Amniotic fluid levels of soluble VEGFR-1 in women with FGR (median 19.5 ng/mL, range 5-40 ng/mL) did not statistically differ from those of controls . The AF levels of soluble VEGFR-1 did not correlate with those of erythropoietin . Soluble VEGFR-1 was clearly detectable (median 14 ng/mL, range 9-22 ng/mL) in culture media from placental biopsies (n = 20) . CONCLUSION: Preeclampsia is associated with increased levels of soluble VEGFR-1, which are independent of erythropoietin, another hypoxia-inducible factor.

Dig Dis Sci, 2000 Feb, 45(2), 291 - 7
Spontaneous apoptotic DNA fragmentation in cultured guinea pig gastric mucosal cells; Tsutsumi S et al.; The purpose of this study was to elucidate the mechanism of spontaneous and rapid cell death of cultured gastric pit cells . Gastric pit cells have a rapid cell turnover rate in vivo . We here show that guinea pig gastric pit cells in culture undergo spontaneous and rapid apoptotic DNA fragmentation, which may represent the rapid cell turnover cycle of gastric pit cells in vivo . This spontaneous apoptotic DNA fragmentation required the presence of fetal calf serum in the culture media . Furthermore, the spontaneous apoptotic DNA fragmentation was prevented by protein synthesis and caspase inhibitors.

Anim Reprod Sci, 2000 Mar 15, 58(3-4), 229 - 40
The effect of macromolecular supplementation on the surface tension of TCM-199 and the utilization of growth factors by bovine oocytes and embryos in culture; Palasz AT et al.; The objectives of this study were to determine the surface tension of bovine follicular fluid (BFF) and TCM-199, and the effects of the synthetic surfactant, Twin-80, or FCS, in TCM-199 on surface tension measurements and subsequent effects on bovine oocyte and embryo development . The surface tension of BFF was determined to be approximately 45.5 mN m(-1) at 25 degrees C and approximately 42.7 mN m(-1) at 39 degrees C, which was comparable to the surface tension of TCM-199 containing Twin-80 (45.7 and 43.2 mN m(-1), respectively) . There was no difference in surface tension measurements of BFF from follicles 2-7 mm or 8-15 mm in diameter . Both Millipore water and phosphate buffered saline (PBS) had a surface tension measurement of 69.5 mN m(-1) at 39 degrees C . Although the presence of Twin-80 in TCM-199 resulted in a reduction in surface tension measurement as compared with unsupplemented TCM-199, there was no effect on the number of oocytes reaching metaphase II . However, the addition of epidermal growth factor (EGF) to TCM-199 containing Twin-80 did result in increased maturation rates of oocytes in vitro . There was no effect of insulin, transferrin, and selenium (ITS), or EGF on surface tension measurement of TCM-199, but significantly more zygotes cleaved and developed to morulae/blastocysts in TCM-199 containing both Twin-80 and growth factors . The addition of 20 microg EGF ml(-1) to TCM-199 containing Twin-80 was as efficacious in supporting bovine embryos in culture as was the addition of 5% or 10% fetal calf serum . This study demonstrates that surface-active components in culture media positively affect bovine oocyte maturation and embryo development in culture . Data also suggest that non-ionic surfactants, such as Twin-80 in TCM-199, may successfully replace the surface-active properties but not the embryotrophic properties of serum in embryo maturation/culture media . Although commercial TCM-199 (containing Twin-80) did not require the addition of other surface-active compounds to lower surface tension, it did benefit from the addition of growth promoting factors, which were also provided by serum.

FEBS Lett, 2000 Mar 3, 469(1), 1 - 4
Expression of type XVI collagen in cultured skin fibroblasts is related to cell growth arrest; Tajima S et al.; The expression of type XVI collagen in various phases of cell growth in cultured skin fibroblasts was studied . A marked increase in type XVI collagen mRNA level was found in stationary phases of cell growth (non-adherent and confluent phases), whereas the expression of type I and III collagens was undetectable in the non-adherent phase but became greater in the confluent phase . When suspended cells were further cultured over 72 h (suspension arrest), mRNA level and gene transcription of type XVI collagen were time-dependently increased whereas those of type I collagen remained undetectable . When the confluent cells were further cultured for 72 h under the condition of serum deprivation (serum deprivation arrest), mRNA levels of both type XVI collagen and type I collagen were elevated . The level of type XVI collagen polypeptide in the culture media of suspension-arrested and serum deprivation-arrested cells paralleled the mRNA level of type XVI collagen . The results indicate that expression of type XVI collagen (a member of the fibril-associated collagens with interrupted triple helices), unlike interstitial collagens (type I collagen), is related to cell growth arrest brought about by two different growth inhibiting systems, suspension arrest and serum deprivation arrest.

Biochem Biophys Res Commun, 2000 Mar 16, 269(2), 532 - 6
Cancer cells responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation; Nagai M et al.; A novel cDNA encoding a secreted form of osteoclast differentiation factor/tumor necrosis factor-related activation-induced cytokine (sODF/TRANCE, GenBank Accession No . AB037599) was sequenced from 5' RACE cDNA clones of squamous cell carcinoma cell lines, SCC-4 and T3M-1 Cl.2, of which parental malignant tissues had caused severe humoral hypercalcemia . The sODF/TRANCE cDNA was composed of unknown 5' end sequence followed by the 100% identical sequence of the ODF/TRANCE extracellular domain-coding region . The longest open reading frame (ORF) of the novel cDNA completely matched the 3' end of the ORF of the ODF/TRANCE cDNA encoding C-terminal amino acid residues (74-318) in the extracellular region . The corresponding protein that reacted with the antibody specific for the extracellular domain of ODF/TRANCE was detected in the culture media conditioned by the cancer cells . Furthermore, human promyeloblastic leukemia cells, HL60, differentiated into osteoclast-like cells (OCLs) when cultured in the media conditioned by SCC-4 and T3M-1 Cl . 2 cells . The differentiation of HL60 cells into OCLs was inhibited by the anti-ODF/TRANCE antibody . These results strongly suggest that sODF/TRANCE plays an important role in enhanced bone-resorption in humoral hypercalcemia of malignancy .

Cancer Res, 2000 Feb 15, 60(4), 1014 - 20
Apoptosis induction of human myeloid leukemic cells by ultrasound exposure; Ashush H et al.; Therapeutic ultrasound (ULS) and the resulting cavitation process has been shown to induce irreversible cell damage . In this study, we wanted to further investigate the mechanism of ULS-induced cell death and to determine whether apoptosis is involved . High intensity focused pulsed ULS sonication at a frequency of 750 KHz was delivered to HL-60, K562, U937, and M1/2 leukemia cell line cultures . ULS exposure used with induction of transient cavitation in the focal area was delivered with an intensity level of 103.7 W/cm2 and 54.6 W/cm2 spatial-peak temporal-average intensity . As a control, ULS of lower intensity was delivered at 22.4 W/cm2 spatial-peak temporal-average intensity, presumably without generation of cavitation . Our results indicated that DNA damage induced by ULS cavitation did not involve generation of free radicals in the culture media . Morphological alterations observed in cells after exposure to ULS included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation . Apoptotic cells were evaluated by fluorescence microscopy and detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which identifies DNA breaks, and by the leakage of phosphatidylserine from the inner to the outer side of the membrane layer of treated cells . Some bioeffects induced on sonicated HL-60 cells, such as inhibition of cell proliferation, DNA repair, and cell-dependent apoptosis, were found to be similar to those produced by gamma-irradiation . Thus, much of the cell damage induced by therapeutic ULS in leukemia cells surviving ULS exposure appears to occur through an apoptotic mechanism.

Int J Pharm, 2000 Mar 20, 197(1-2), 161 - 8
In vitro release studies of methylmethacrylate liberation from acrylic cement powder; Bettencourt A et al.; Bone cement or polymethylmethacrylate (PMMA) is commonly used for anchoring cemented prosthesis to the bone . Cytotoxic effect of culture media exposed to PMMA powder may be related with long term problems associated with acrylic cement application, being the monomer (methylmethacrylate) one of the cement's component partly responsible for the cytotoxic effect . The present work reports the studies of monomer release from acrylic bone cement powder under different experimental conditions: setting time of PMMA (in solution and air) and different culture media composition . High-performance liquid chromatography was used for the determination of residual monomer . Mathematical models were applied to experimental dissolution data revealing that monomer release is lightly affected by the studied variables . The monomer release seems to be a surface phenomena, suggesting that the possible actions of monomer will mainly be due to the initial loss of non polymerized monomer rather than to further depolymerization of the already polymerized cement.

Domest Anim Endocrinol, 2000 Jan, 18(1), 127 - 32
Oxidized-low density lipoprotein inhibits cyclic AMP production by porcine luteal cells; Brannian JD et al.; Oxidized(OX)-low density lipoprotein (LDL) inhibits steroidogenesis by luteal cells (LC) from regressing porcine CL . The present study was designed to investigate the mechanism of inhibition by determining whether OX-LDL inhibits basal and agonist-stimulated cAMP production in regressing LC . Collagenase-dispersed porcine LC (n = 7 animals, estrous cycle Day 12-15) were cultured (2.5 x 10(5) cells/0.5 ml) in serum-free DMEM/Hams F-12 in duplicate wells at 37 degrees C . Approximately 18 hr after plating, media were replaced and LC were immediately treated with human LDL (0, 25, or 100 microg/ml) or OX-LDL (25 or 100 microg/ml) . LC were incubated for 2 hr before addition of isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity, immediately followed by hCG (100 ng/ml), cholera toxin (CT; 0.1 microM), forskolin (FS; 50 microM), or no further treatment (controls) . LC were incubated for an additional 90 min . After removal of culture media, cells were extracted with 0.1 N HCl . Cell extracts were assayed for cAMP by enzyme immunoassay (EIA) . HCG, CT, and FS increased (P < 0.05) cAMP production approximately four-, 10-, and 25-fold, respectively, relative to controls . OX-LDL (25 and 100 microg/ml) inhibited (P < 0.05) cAMP production by unstimulated, hCG-, and CT-stimulated LC, but not that by FS-stimulated LC . The highest concentration of OX-LDL (100 microg/ml) reduced cAMP formation by 39.8 +/- 6.6%, 44.7 +/- 10.5%, and 67.7 +/- 4.5% in unstimulated, hCG-, and CT-stimulated LC, respectively . In contrast, unmodified LDL (25 and 100 microg/ml) did not alter cAMP production . We conclude that OX-LDL can interfere with the cAMP signaling pathway in regressing luteal cells by acting at sites proximal to adenylate cyclase activation.

Anim Reprod Sci, 2000 Feb 28, 58(1-2), 113 - 25
Response of porcine theca and granulosa cells to GH during short-term in vitro culture; Gregoraszczuk EL et al.; In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested . In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts . In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied . Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH . Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures . The media were assayed after 48 h of culture for progesterone and oestradiol by RIA . GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles . A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed . GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles . Both co-culture systems exhibited synergistic effect on oestradiol secretion . The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone . In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation . A statistically significant increase in oestradiol secretion was observed in all culture conditions . The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells . In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.

Anim Reprod Sci, 2000 Feb 28, 58(1-2), 87 - 98
The role of luteal steroid hormones in the regulation of the estrous cycle of high fecundity Olkuska sheep; Zi&ecedil;ba DA et al.; The study was undertaken to investigate the steroid hormone production by sheep luteal cells . Corpora lutea were collected from 30 Olkuska sheep on Days 3, 6, 9, 12 and 15 of the estrous cycle during the reproductive season . In Experiment 1, steroid hormone concentration was estimated in extracts of CL . In Experiment 2, luteal cells were cultured in vitro for 24 h . Luteal cells isolated on Days 9 and 12 secreted high amounts of progesterone and androgens but smaller amounts of estradiol . Concentration of these steroids in CL extracts collected on the same days showed the same trend . In CL harvested on Day 15, a decrease in androgens and progesterone as well as a significant increase in estradiol were observed in culture media and in extracts . Judging from the high amounts of estradiol and low amounts of androgen observed at the end of the luteal phase, we speculate that the steroid hormones secreted by the regressing CL may play an active role in the regulation of the estrous cycle in the Olkuska sheep with autocrine influence on the luteal activity or a possible paracrine action on follicular growth.In the third Experiment, the possibility of heterogeneity in the multiple corpora lutea population of prolific Olkuska sheep was investigated . Differences were found in the level of progesterone and estradiol secretion by individual corpora lutea recovered from the same animal, which also varied in terms of weight . This is the first study which shows the existence of intra-ovarian and individual heterogeneity between corpora lutea recovered from ewes during the normal estrous cycle.

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Last modified: May 25, 2005