Br J Pharmacol . 2005 Jan 17; {Epub ahead of print} The pore-forming subunit of the K(ATP) channel is an important molecular target for LPS-induced vascular hyporeactivity in vitro; O'brien AJ et al.; ATP-sensitive K(+) (K(ATP)) channel activation is implicated in the vascular hyporeactivity occurring in septic shock . However, channel inhibition with the sulphonylurea receptor (SUR) antagonist, glibenclamide (Glib) fails to reverse lipopolysaccharide (LPS)-induced vascular hyporeactivity in vitro . We investigated whether inhibitors that act by binding to the K(ATP) channel pore could be effective . Ring segments of endothelium-intact rat mesenteric artery were incubated with LPS in culture media for either 6 or 20 h before contractile responses to phenylephrine were assessed in the absence or presence of K(ATP) channel inhibitors . The pore-forming subunit inhibitors barium chloride (BaCl(2); 300 muM) and PNU-37883A (1 muM) significantly reversed hyporeactivity at both time points, although less so at 20 h . In contrast, the SUR inhibitors, Glib (10 muM), tolbutamide (Tolb) (1 mM) and PNU-99963 (1 muM) were ineffective . In LPS-incubated tissues, Glib and Tolb antagonised contractions to the thromboxane A2 mimetic, U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha)) (10(-7) M), whereas the pinacidil-derived inhibitor, PNU-99963, did not.Contractions to 60 mM KCl were unaffected by LPS at 6 h, but were significantly depressed by LPS at 20 h, suggesting that K(+)-channel-independent pathways contribute to hyporeactivity at the later time point.The inducible nitric oxide synthase (iNOS) inhibitor, 1400 W (10 muM) and Tolb inhibited the production of nitrite induced by LPS, whereas BaCl(2) and PNU-37883A had no effect.In conclusion, K(ATP) channels contribute to LPS-induced vascular hyporeactivity via the iNOS pathway in rat mesenteric artery . The effectiveness of pore inhibitors over SUR inhibitors of the K(ATP) channel suggests altered SUR function following LPS administration, which cannot be explained by thromboxane receptor inhibition.British Journal of Pharmacology advance online publication, 17 January 2005; doi:10.1038/sj.bjp.0706065. Bioprocess Biosyst Eng . 2005 Jan 15; {Epub ahead of print} MIR spectroscopic analysis on sugar metabolic and ethanol productive kinetics of suspension TBY-2 and rice cells pre-cultured in various media; Yamanaka A et al.; The influence of sugars in pre-cultivation media suspended plant cells on the kinetics of the sugar uptake and the ethanol production was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR) . We performed the plant cell cultivation with Nicotiana tabacum cv . Bright Yellow No.2 (TBY-2) cells and Oryza sativa L., Japonica, cv . Nipponbare (rice) cells, respectively, in pre-culture and culture media, which had various types of glucose, fructose, sucrose or glucose-fructose mixtures . The results confirmed the kinetic differences between the TBY-2 cells and rice cells . These results suggested that the TBY-2 cells consumed sugar before growth and the rice cells consumed sugar after growth, moreover, the ethanol content increased just after cell growth was activated based on the non-dimensional cultivation time for the cell growth behavior. Blood Coagul Fibrinolysis, 2005 Jan, 16(1), 9 - 16 Genetic analysis of hereditary factor X deficiency in a French patient of Sri Lankan ancestry: in vitro expression study identified Gly366Ser substitution as the molecular basis of the dysfunctional factor X; Isshiki I et al.; We investigated a new family with cross-reactive material-positive factor X (FX) deficiency . The proband was an 11-year-old French girl from Sri Lanka with a tendency towards severe bleeding . The FX antigen level was 67%, although the activity with extrinsic pathway was 1 U/dl . The complete nucleotide sequences of all exons and exon/intron junctions of the patient's genomic DNA revealed a homozygous G <-- A substitution in exon 8, which would result in replacement of Gly366 with Ser . The proband is the first reported case of homozygote for the FX Gly366Ser mutation . Heterozygosity for Gly366Ser substitution was previously reported in a Japanese patient (FX Nagoya 2) . We studied the functional consequences by expressing mutant FX Gly366Ser protein in HEK293 cells . FX Gly366Ser was secreted into the culture media at levels similar to wild-type FX; however, mutant FX activities were only 0.04, 1.05, and 0.75% of wild-type FX upon activation by the extrinsic system, the intrinsic system, and Russell's viper venom, respectively . Moreover, the activity of FX Gly366Ser was undetectable when analyzed with chromogenic-activated FX and thrombin generation assays . These data suggest that the Gly366Ser substitution would cause a major defect in function of the FX molecule. Mol Cell Biochem, 2004 Nov, 266(1-2), 17 - 24 AGE-R3/galectin-3 expression in osteoblast-like cells: regulation by AGEs; Mercer N et al.; The accumulation of irreversible advanced glycation endproducts (AGEs) on long-lived proteins, and the interaction of AGEs with cellular receptors such as AGE-R3/galectin-3 and RAGE, are considered to be key events in the development of long-term complications of diabetes mellitus, Alzheimer's disease, uremia and ageing . The aim of this study was to investigate the expression and sub-cellular distribution of galectin-3, as well as its possible modulation by AGEs, in MC3T3E1 mouse calvaria-derived osteoblasts and in UMR 106 rat osteosarcoma cells . Both osteoblastic lines were cultured either with control bovine serum albumin (BSA) or with AGEs-BSA for 48 h . Cells were evaluated for galectin-3 expression by fixing and immunofluorescent microscopic analysis; or Western blot analysis of whole cell extracts, sub-cellular fractions and culture media . Both cell lines express 30 kDa (monomeric) galectin-3, although expression was about 15-fold lower in the UMR106 osteosarcoma cells . Dimeric (70 kDa) galectin-3 was additionally observed in the UMR106 cells . Immunofluorescent analysis of galectin-3 distribution showed a diffuse cytoplasmic and strong nuclear pattern in MC3T3E1 osteoblasts, and a patchy cytoplasmic pattern in UMR106 cells . Western blot analysis for both cell lines showed that galectin-3 was mainly found in the cytoplasm and in minor amounts in the microsomal fraction, while considerable amounts were secreted into the culture media . Exposure to 100-200 microg/mL AGEs-BSA increased the cellular content of 30 kDa galectin-3 (20-25% for MC3T3E1 and 35-70% for UMR106 versus control BSA, p < 0.05), and decreased the culture media levels of galectin-3 (10-20% for MC3T3E1 and for UMR106 versus control BSA, p < 0.05) . These results confirm the expression of galectin-3 in osteoblastic cells, and suggest different levels and sub-cellular distribution of this protein in transformed versus non-transformed osteoblasts . Osteoblastic exposure to AGEs alters their expression and secretion of galectin-3, which could have significant consequences on osteoblast metabolism and thus on bone turnover. Arthritis Rheum, 2005 Jan, 52(1), 128 - 35 Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage; Kobayashi M et al.; OBJECTIVE: To determine whether interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha), or both, plays a role in the excessive degradation that is observed in cultured osteoarthritic (OA) articular cartilage . METHODS: Antagonists of IL-1 and TNFalpha, namely, IL-1 receptor antagonist and the PEGylated soluble TNFalpha receptor I, respectively, were added at different concentrations to explant cultures of nonarthritic (5 obtained at autopsy) and OA (15 obtained at arthroplasty) articular cartilage . The cleavage of type II collagen (CII) by collagenase was measured by an immunoassay in cartilage and culture media . Proteoglycan (mainly aggrecan) content and degradation were measured by a colorimetric assay for glycosaminoglycan (GAG) content in cartilage and culture media . Reverse transcriptase-polymerase chain reaction was used to analyze gene expression of matrix metalloproteases (MMPs) 1, 3, and 13, CII, aggrecan, IL-1, and TNFalpha . RESULTS: Antagonists of IL-1 and TNFalpha inhibited the increase in CII cleavage by collagenase as well as the increase in GAG release observed in OA cartilage compared with normal cartilage . Inhibition was significant in tissue from some patients but not from others, although significant inhibition was observed when all the results were analyzed together . An increase in the GAG content in cartilage was seen in 4 of 15 cases . However, this increase was not significant when all the data were combined . Preliminary results indicated no effect of these antagonists on nonarthritic cartilage from 3 different donors . Independent analyses of gene expression in cultured cartilage from 9 other OA patients revealed that IL-1 or TNFalpha blockade, either alone and/or in combination, frequently down-regulated MMP-1, MMP-3, and MMP-13 expression . Expression of IL-1 and TNFalpha was inhibited by either antagonist or by the combination in essentially half the cases . The combined blockade up-regulated aggrecan and CII gene expression in approximately half the cases . CONCLUSION: These results suggest that the autocrine/paracrine activities of TNFalpha and IL-1 in articular cartilage may play important roles in cartilage matrix degradation in OA patients but not in all patients . Inhibition of either or both of these cytokines may offer a useful therapeutic approach to the management of OA by reducing gene expression of MMPs involved in cartilage matrix degradation and favoring its repair. Arthritis Rheum, 2005 Jan, 52(1), 84 - 93 Interleukin-6 receptor shedding is enhanced by interleukin-1beta and tumor necrosis factor alpha and is partially mediated by tumor necrosis factor alpha-converting enzyme in osteoblast-like cells; Franchimont N et al.; OBJECTIVE: Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions . The goal of the present study was to investigate changes in sIL-6R levels in response to the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) and to determine the role of TNFalpha-converting enzyme (TACE) in this process . METHODS: Levels of sIL-6R in the culture media of MG63 and SAOS-2 osteoblast-like cell lines after exposure to various agents were determined by immunoassay . TACE protein levels were measured by Western immunoblotting . Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL-6R and TACE to determine the potential involvement of TACE in IL-6R shedding . RESULTS: IL-1beta and TNFalpha increased the levels of sIL-6R in the culture media of MG63 osteoblast-like cells . This effect was not influenced by cycloheximide or 5,6-dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156) . IL-1beta and TNFalpha had no influence on the alternatively spliced form of IL-6R RNA . Levels of sIL-6R were reduced when MG63 cells were transiently transfected with TACE siRNA . Transfection of SAOS-2 cells with expression plasmids for IL-6R and TACE produced a dose-dependent increase in sIL-6R levels . CONCLUSION: IL-1beta- and TNFalpha-mediated induction of IL-6R shedding in osteoblast-like cells is at least partly dependent on TACE activation. Clin Immunol, 2005 Feb, 114(2), 147 - 53 Exogenous leptin restores in vitro T cell proliferation and cytokine synthesis in patients with Common Variable Immunodeficiency Syndrome; Goldberg AC et al.; Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia . Leptin has been implicated as an antiapoptotic compound as well as a stimulant of the immune response . Leptin administration is capable of reversing the immune deficiency that occurs upon starvation . We investigated a possible role for leptin in CVID; a condition associated with lowered plasma leptin levels . Thirty-eight patients were studied . Addition of leptin to the tissue culture media of PBMC from CVID patients increased the proliferative response of lymphocytes to mitogens and decreased activation-induced apoptosis of these cells . IL-2 and specially IL-4 production also increased significantly upon addition of leptin to the PBMC cultures . Our results suggest that leptin may be involved in some of the cellular defects observed in CVID and indicate a novel therapeutic strategy to improve immune function in these patients. Zhonghua Fu Chan Ke Za Zhi, 2004 Nov, 39(11), 747 - 9 {Effect of luteinized granulosa cell conditioned medium on cortical granule of mouse oocytes matured in vitro.}; Bai XH et al.; OBJECTIVE: To study the effect of luteinized granulosa cell conditioned medium on cortical granule (CG) of the mouse oocytes matured in vitro . METHODS: Oocytes in germinal vesicle (GV) stage of Kunming mice were randomly divided into 2 groups according to different in vitro maturation (IVM) culture media . The study group medium contained 50% granulosa cell condition medium, follicle stimulating hormone 75 U/L and estrodial 1 nmol/L . The control group medium contained follicle stimulating hormone 75 U/L and estrodial 1 nmol/L . Oocytes were cultured for 16 or 18 hours . CG was examined by fluorescein isothiocyanate labeled Lens culinaris agglutinin under a confocal scanning laser microscope . RESULTS: After cultured for 16 hours, the nuclear maturation rates of control and study groups were 70.0% and 76.5% . After cultured for 18 hours, the maturation rates were 75.1% and 83.1%, respectively . There was no significant difference between the two groups . After cultured for 16 hours, there was no pronuclear formation in both groups . When culture was extended to 18 hours, fertilization occurred . After cultured for 16 hours, the rates of CGs forming a line under membrane were 10.0% and 50.0% in control and study groups respectively . When culture was extended to 18 hours, the rates rose to 57.1% and 91.6% accordingly . The rate of 18 h of each group was significantly higher than that of 16 h (both P < 0.001) . The rate of study group of 18 h was significantly higher than that of control group (P < 0.05) . CONCLUSION: Granulosa cell conditioned medium could improve the mouse oocytes maturation competence in vitro. Biomed Mater Eng, 2005, 15(1-2), 51 - 63 Renal epithelia in long term gradient culture for biomaterial testing and tissue engineering; Minuth WW et al.; In the organism epithelia perform perfect barrier functions . Strong rheological and mechanical influences constitute the normal environment of this tissue throughout life . Most epithelia are exposed to different fluids at the luminal and basal sides . To obtain realistic information about tissue development in modern biomaterial testing and tissue engineering it is necessary to mimik the natural environment of epithelia . Cultured cells are brought in contact with an artificial extracellular matrix to determine whether proper development into a functional epithelium occurs . As under natural conditions the cultures have to withstand mechanical and fluid stress over a prolonged period of time in close contact to a selected biomaterial . However, development of tissue-specific features such as polarization, tightness and transport under in vitro conditions will only occur, if the biomaterial and the culture conditions support tissue development . Leakage, edge damage and pressure differences during culture have to be avoided so that the natural functions of the growing epithelium can develop . Our aim is to generate functional epithelia derived from renal explants containing stem cells, which are microsurgically isolated and placed into specific O-ring carriers for optimal handling . The cells develop in combination with a collagenous matrix from an embryonic into a functional collecting duct (rCD) epithelium . To achieve optimal culture conditions the tissue is placed in a gradient culture container . A typical environment can be simulated by superfusing different culture media at the luminal and basal sides . Within days epithelia growing inside the gradient container build up a physiological barrier, which is maintained during the whole culture period . The described method allows to investigate the influence of new biomaterials over prolonged periods of time. Indian J Exp Biol, 2004 Dec, 42(12), 1235 - 8 In vitro explant culture of mantle epithelium of freshwater pearl mussel; Barik SK et al.; The in vitro culture of nacre secreting pallial mantle explants of freshwater pearl producing mussel, Lamellidens marginalis (Lamarck) included depuration of pearl mussels with different physical and chemical agents to eradicate various commensals, removal of pallial mantle ribbon, aseptic preparation of explants from the ribbon and transfer of those explants into tissue culture petri dishes . Special synthetic tissue culture media enriched with additives viz., inactivated calf fetal serum and antibiotics were poured into plates with explants . The culture plates were incubated at 30 degrees C in a CO2 incubator at 5%, CO2 . The cultures could be maintained for 42-45 days without any contamination . After 12 hr epithelial like cells began to migrate out and formed a complete cell sheet surrounding the explant within 12-15 days . The epithelial cells in the culture indicated functional viability as subsequently after 38-40 days of culture, typical aragonitic 'nacre' crystals of CaCO3 could be observed throughout the culture plates. Ann Surg, 2005 Jan, 241(1), 125 - 33 Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function; Sosef MN et al.; OBJECTIVE: To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution . SUMMARY BACKGROUND DATA: Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes . METHODS: Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO . Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days . Hepatocyte yield and viability were assessed up to 14 days postthaw . Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed . RESULTS: Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%) . Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%) . Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls . CONCLUSIONS: This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge . These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices. FEMS Microbiol Lett, 2005 Jan 1, 242(1), 45 - 50 Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa; Kumar KS et al.; Two metal response elements, flanking an antioxidant response element, were identified in regions upstream (-3730 bp) to copper metallothionein (CuMT) gene of Neurospora crassa . Presence of copper in culture media, but not of pro-oxidants like H(2)O(2) or menadione, induced CuMT gene expression that could not be completely abolished by antioxidants such as N-acetyl cysteine and ascorbic acid . Gel shift assays revealed the ability of nuclear extracts from copper induced cultures to bind PCR-amplified metal response or antioxidant response elements . Similar observations could not be made with cultures exposed either to pro-oxidants or antioxidants . These results differentiate between CuMT gene induction by copper from antioxidant functions associated with the identified upstream elements. Cell Mol Biol (Noisy-le-grand) . 2004 Dec 24;Suppl.50:OL701-OL712 {Epub ahead of print} ENDOTHELIN-2 DOWN-REGULATION OCCURS IN PARALLEL WITH THE ANTI-PROLIFERATIVE EFFECT OF DIMETHYLSULFOXIDE IN BeWO HUMAN CHORIOCARCINOMA CELL LINE; Rebourcet R et al.; Endothelins exhibit growth regulating properties in many cell types, and there is now considerable evidence that they play a critical pathophysiological role in human diseases such as carcinogenesis . In the choriocarcinoma cell lines JEG-3, JAR and BeWO, we demonstrate by RT-PCR that prepro endothelin (ET)-1 and prepro ET-2 mRNA were expressed, whereas prepro ET-3 was never expressed . Only ET-1 and ET-2 peptides were identified by HPLC/RIA analyses in culture media from these three choriocarcinoma cell lines . In the BeWO line, the cellular growth measured as the cell count and DNA content decreased with increasing concentrations of dimethyl sulfoxide (DMSO; range 0.5-3%) . The expression of prepro ET-2 was also suppressed by DMSO, whereas no change was noticed in prepro ET-1 mRNA . All these effects were reversible when DMSO was replaced by 15% foetal calf serum . These effects of DMSO which are correlated to ET-2 down regulation in dividing BeWO cells suggest a role for this endothelin isoform in trophoblast proliferation. Lab Chip, 2005 Feb, 5(1), 56 - 63 Epub 2004 Jul 22. Perfusion and chemical monitoring of living cells on a microfluidic chip; Shackman JG et al.; A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans . In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 {micro sign}L min(-1) . The flow was driven by gas pressure applied off-chip . Perfusate was continuously sampled at 2 nL min(-1) by electroosmosis through a separate channel on the chip . The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel . The cell chamber and reaction channel were maintained at 37 {degree}C . The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V cm(-1) . The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions . Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose . The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM . The 2.5 cm {times} 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion . The technique should be amenable to studies involving other tissues or cells that release chemicals. Wei Sheng Yan Jiu, 2004 Sep, 33(5), 552 - 4 {Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry used to bacteria detection}; Sun Z et al.; To investigate the influence variations, three culture media and different culture time were evaluated using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the rapid identification of eight strains of bacteria . These culture media are Colombia Blood Agar, Eosin Ethylene Blue Agar and Nutrition Agar . Results demonstrate that different culture media and culture time can influence the reproducibility of the bacteria fingerprint . It is suggested that appropriate culture media and culture time can enhance the accuracy of the bacteria detection using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Microbiol Immunol, 2004, 48(12), 985 - 94 Quantum dots targeted to the assigned organelle in living cells; Hoshino A et al.; Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes . Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells . We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands . When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation . In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells . In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo. Pediatr Res . 2004 Dec 20; {Epub ahead of print} Effects of Maternal Starvation on Hepatocyte Proliferation in the Late Gestation Fetal Rat; Gruppuso PA et al.; Fetal growth retardation, a common end point for a variety of conditions affecting mother and fetus, is associated with reduced liver mass . We have performed studies to determine the mechanism for decreased liver mass in a maternal starvation model of fetal growth restriction in the rat . Pregnant dams were deprived of food for 48 h before delivery on embryonic day 19 (E19) . Fetal body weight was not affected . However, fetal liver weight was reduced by approximately 15% . Immunostaining of fetal liver for proliferating cell nuclear antigen and flow cytometry on isolated fetal hepatocytes showed G1 cell cycle arrest in samples from starved dams . Based on our prior studies showing attenuated hepatic insulin signaling in the late gestation fetal rat, we tested the hypothesis that G1 arrest in our model might be due to altered nutrient signaling . Fetal plasma amino acid analyses showed no decrease in branched-chain amino acids, but arginine concentrations were decreased in fetuses of fasted mothers . Reduced arginine in E19 fetal hepatocyte culture media was associated with decreased DNA synthesis . Whereas levels of cyclins D and E were unchanged in fetal hepatocytes exposed to low arginine, cyclin E-dependent kinase activity was reduced . Low arginine also induced changes in the translational machinery, indicative of impaired signaling through the nutrient sensing kinase mammalian target of rapamycin . Our results are consistent with the hypothesis that restricted nutrient availability signals to the hepatocyte cell cycle in fetuses of fasted mothers, thereby accounting for decreased hepatocyte proliferation and liver mass. J Invest Dermatol, 2004 Dec, 123(6), 1012 - 9 Heat shock-induced matrix metalloproteinase (MMP)-1 and MMP-3 are mediated through ERK and JNK activation and via an autocrine interleukin-6 loop; Park CH et al.; Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on skin aging . In addition to photons, heat is likely to be generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stress . Here we investigated the effect of heat shock on the expressions of matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 in cultured human skin fibroblasts . Heat shock induced the expression of MMP-1 and MMP-3, but not MMP-2, at the mRNA and protein levels in a temperature-dependent manner, and caused the rapid activation of three distinct mitogen-activated protein kinases (MAPK), extracelluar signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK . The heat shock-induced MMP-1 and MMP-3 expression was suppressed by the inhibition of ERK and JNK but not by p38 MAPK inhibition . Furthermore, heat shock increased the synthesis and release of interleukin-6 (IL-6) into culture media . The specific inhibition of IL-6 using a monoclonal antibody against IL-6 greatly reduced the expression of MMP-1 and MMP-3 induced by heat shock . Taken together, our results suggest that ERK and JNK play an important role in the induction of MMP-1 and MMP-3 by heat shock and that the heat shock-induced expression of MMP-1 and MMP-3 is mediated via an IL-6-dependent autocrine mechanism. J Org Chem, 2004 Dec 24, 69(26), 8987 - 96 Synthesis and characterization of a cobalamin-colchicine conjugate as a novel tumor-targeted cytotoxin; Bagnato JD et al.; Colchicine was derivatized at C7 with p-alkoxyacetophenone and conjugated to cobalamin (vitamin B(12)) through an acid-labile hydrazone linker . The cobalamin moiety leads to preferential uptake of the cobalamin-colchicine prodrug by cancer cells, whereupon the hydrazone linker undergoes hydrolysis in the lysosome to unmask colchicine, which acts as a potent cytotoxin by stabilizing microtubules and causing cell death . The bioconjugate is stable in cell culture media and at neutral pH but undergoes hydrolysis with a half-life of 138 min at pH 4.5 . The colchicine-cobalamin bioconjugate exhibits nanomolar LC(50) values against breast, brain, and melanoma cancer cell lines in culture . Attachment of colchicine to cobalamin is expected to increase the therapeutic index of the drug by limiting the side effects caused by the current nonselective administration of tubulin-targeted chemotherapeutic drugs. Am J Physiol Regul Integr Comp Physiol . 2004 Dec 16; {Epub ahead of print} Adiponectin inhibits LPS-induced NFkB activation and IL-6 production, and increases PPAR{gamma}2 expression in adipocytes; Ajuwon KM et al.; Circulating adiponectin concentrations are lower in association with obesity and insulin resistance, both of which are physiological conditions frequently accompanied by chronic increases in circulating interleukin-6 (IL-6) and(or) tumor necrosis factor-alpha (TNFalpha) . Adiponectin suppresses activation of the nuclear factor kappa B (NFkB) transcription factor in aortic endothelial cells and porcine macrophages . We hypothesized that adiponectin acts as an anti-inflammatory hormone and suppresses the activation of NFkB, which culminates in increased cytokine production by adipocytes and other immune cells . Because PPARgamma2 exerts anti-inflammatory actions and antagonizes the transcriptional activity of NFkB, we also determined if adiponectin alters PPARgamma2 expression in pig adipocytes . Additionally, we determined whether interferon-gamma (IFNgamma) altered the expression of PPARgamma2 adiponectin in the presence or absence of adiponectin . Primary pig adipocytes were recovered from subcutaneous adipose tissue, and incubated in the presence and absence of lipopolysaccharide (LPS, 10 microg/mL) and adiponectin (30 microg/mL), and nuclear extracts recovered for electrophoretic mobility shift assays to assess nuclear localization of NFkB . Whereas LPS caused a marked increase in nuclear NFkB, adiponectin attenuated the response, and also attenuated the induction of IL-6 expression by LPS (P < 0.05) . We extended this work to 3T3-L1 adipocytes, and obtained similar results . Additionally, adiponectin antagonized the endotoxin-induced increase in TNFalpha mRNA expression (P < 0.05) and tended (P < 0.065) to diminish the accumulation of this cytokine in the culture media in 3T3-L1 adipocytes . Incubation with adiponectin (30 microg/mL) resulted in a marked upregulation of PPARgamma2 mRNA (P < 0.05) within 5 hours . Although IFNgamma did not reduce the basal expression of PPARgamma2, it did suppress the induction of this PPARgamma by adiponectin (P < 0.05) . These data indicate that adiponectin is a local regulator of inflammation in the adipocyte and adipose tissue, and that the mechanism includes regulation of the NFkB transcription factor, and possibly an induction of PPARgamma2. Environ Sci Pollut Res Int, 2004, 11(6), 388 - 93 Reaction of spruce cells toward heavy metals and the influence of culture conditions; Schroder P et al.; BACKGROUND: Plant cell cultures may serve as biosensors for the detection of heavy metals and other toxic substances . Standard culture media and protocols are frequently utilised, but in these media no care is usually taken to control the influence of hormones and nutrients on the reaction of the enzymes or m under consideration as parts of the sensor . The present paper investigates the influence of media composition on the reaction of spruce cells towards heavy metals . METHODS: Spruce cell cultures were grown in a standard medium, either i) alone, ii) containing 0.3% sucrose or iii) containing 3% sucrose and the hormones BAP and NAA . The cell cultures were then incubated in medium with fungal elicitor, H2O2, CdSO4 (50 to 500 microM), or, alternatively, with a standard heavy metal mixture containing 80 microM Na2HAsO4, 150 microM CdSO4 and 200 microM PbCl2 . RESULTS: Depending on the nutrient status and hormone availability, large differences in glutathione contents and the GSH/GSSG ratio were observed . However, the cellular redox state seemed to remain more or less constant . Glutathione S-transferase activity was determined with four substrates, and high induction rates for the conjugation of three substrates were observed when hormones were omitted from the media . 1,2-epoxy-nitrophenoxy-propane conjugation was highest in starving cell in the presence of hormones, showing a transient GST induction, with highest rates occurring after 16 hrs following incubation; the induction effect was lost after 24 hrs . CONCLUSION: A medium containing 3% sucrose and both hormones (BAP and NAA) appears to be most favourable for cellular growth as well as the expression of a basis level of detoxification enzymes and antioxidants . With this combination, early responses towards heavy metals at low concentration can be monitored . RECOMMENDATIONS AND PERSPECTIVE: Plant cell cultures are valuable tools for the bioindication of heavy metals and toxic xenobiotics . If standard media and protocols are utilised, the influence of hormones and nutrients on the reaction of the biosensor have to be evaluated thoroughly. J Plant Physiol, 2004 Nov, 161(11), 1245 - 58 Responses of meristematic callus cells of two Cynodon dactylon genotypes to aluminium; Ramgareeb S et al.; Responses to Al3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al3+ toxicity and resistance . A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al3+ ion in culture media, which was supplied as 0.08-2.3 mM Al3+ for 2-8 weeks . Increasing Al3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells . Exposure to 0.8 mM Al3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected . In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis . The Al-R cells appeared to exclude Al3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R) . The results showed that by studying the cellular responses to Al3+ it is possible to discriminate between the Al-S and Al-R C . dactylon genotypes. Hum Reprod, 2005 Jan, 20(1), 49 - 60 Regulation of hepatocyte growth factor by basal and stimulated macrophages in women with endometriosis; Khan KN et al.; BACKGROUND: The different macromolecules as secreted by macrophages (Mvarphi) in the pelvic environment are believed to enhance the growth of endometriosis . However, the possible mediator that stimulates Mvarphi for the production of different growth factors is not well described . Therefore, we investigated the possible production of hepatocyte growth factor (HGF) by the basal and lipopolysaccharide (LPS)-stimulated Mvarphi derived from women with or without endometriosis . METHODS: Using primary culture and 4-well chamber slides, adherent Mvarphi immunoreactive to CD68 were isolated from the peritoneal fluid (PF) of 20 infertile women with endometriosis and 12 women without endometriosis . The proliferation of basal and LPS-treated Mvarphi was investigated by the dimethylthiazole tetrazolioum bromide (MTT) assay . The production of HGF in the culture media of basal and LPS-stimulated Mvarphi was examined by enyme-linked immunosorbent assay . The expression of mRNA for HGF and its receptor, c-Met, in the Mvarphi was investigated by RT-PCR . The effect of HGF on the growth of endometrial cells and Mvarphi was analysed by bromodeoxyuridine (BrdU) incorporation . RESULTS: A >100% increase in the proliferation of peritoneal Mvarphi derived from women with endometriosis, and particularly of those harbouring dominant red lesions, was observed after treatment with LPS (P<0.05) . A 4- and 3-fold increase in the production of HGF was observed by the LPS-treated Mvarphi derived from women with stage I-II endometriosis and stage III-IV endometriosis, respectively, when compared with non-LPS-treated Mvarphi (P<0.001) . At the transcriptional level, we found a 5-fold increase in HGF mRNA expression in LPS-treated Mvarphi versus basal Mvarphi in women with endometriosis (P<0.001) . The BrdU incorporation study indicates that 10-100 ng/ml of HGF enhanced the growth of endometrial epithelial cells, stroma and Mvarphi ( approximately 50% increase) derived from women with endometriosis (all P<0.05) . CONCLUSION: LPS could be an inflammatory mediator of macrophage stimulation in the pelvic microenvironment . Besides mesenchymal cells, HGF is also produced by peritoneal Mvarphi and is possibly involved in the growth of endometriosis. Reprod Domest Anim, 2004 Dec, 39(6), 462 - 7 Different culture media requirements of IVF and nuclear transfer bovine embryos; Mastromonaco GF et al.; Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos . Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos . In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos . In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development . In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development . Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards . Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9 . For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF . These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos. Int J Med Microbiol, 2004 Oct, 294(6), 407 - 12 Comparison of growth of Borrelia afzelii, B . garinii, and B . burgdorferi sensu stricto in MKP and BSK-II medium; Ruzic-Sabljic E et al.; Lyme borreliosis is a tick-borne disease caused by genetically diverse Borrelia strains including B . afzelii, B . garinii, and B . burgdorferi sensu stricto (s.s.) . The aim of the present study was to assess and compare the growth of one strain per species of B . afzelii, B . garinii, and B . burgdorferi s.s . in modified Kelly-Pettenkofer (MKP) and Barbour-Stonner-Kelly-II (BSK-II) medium, and to check for the presence of the overgrowth after inoculating the media with a mixture of two different Borrelia species . All three Borrelia strains grew well in both media . In the majority of the experiments the number of B . afzelii cells was higher in MKP than in BSK-II medium while for B . garinii and B . burgdorferi s.s . a tendency for better growth in BSK-II than MKP was established . In a mixture of equivalent amounts of two species, B . burgdorferi s.s . as a rule overgrew the other two species while in the mixture of B . afzelii and B . garinii the latter was a "dominant" strain . Comparing the performance of the two media, B . burgdorferi s.s . usually overgrew either B . afzelii or B . garinii in MKP as well as in BSK-II medium, however, the results were found to be statistically significant only for MKP medium . In the mixture of B . afzelii and B . garinii the latter was the predominant species but significant differences were established only for BSK-II medium . It seems that the overgrowth is predominantly the result of the characteristics of the individual Borrelia species and most probably not a consequence of growth differences in the two culture media . Further work with a larger number of strains is needed to confirm these findings. Toxicol Appl Pharmacol, 2005 Jan 1, 202(1), 25 - 37 An in vitro biotic ligand model (BLM) for silver binding to cultured gill epithelia of freshwater rainbow trout (Oncorhynchus mykiss); Zhou B et al.; "Reconstructed" gill epithelia on filter supports were grown in primary culture from dispersed gill cells of freshwater rainbow trout (Oncorhynchus mykiss) . This preparation contains both pavement cells and chloride cells, and after 7-9 days in culture, permits exposure of the apical surface to true freshwater while maintaining blood-like culture media on the basolateral surface, and exhibits a stable transepithelial resistance (TER) and transepithelial potential (TEP) under these conditions . These epithelia were used to develop a possible in vitro version of the biotic ligand model (BLM) for silver; the in vivo BLM uses short-term gill binding of the metal to predict acute silver toxicity as a function of freshwater chemistry . Radio-labeled silver ((110m)Ag as AgNO(3)) was placed on the apical side (freshwater), and the appearance of (110m)Ag in the epithelia (binding) and in the basolateral media (flux) over 3 h were monitored . Silver binding (greater than the approximate range 0-100 mug l(-1)) and silver flux were concentration-dependent with a 50% saturation point (apparent K(d)) value of about 10 mug l(-1) or 10(-7) M, very close to the 96-h LC50 in vivo in the same water chemistry . There were no adverse effects of silver on TER, TEP, or Na(+), K(+)-ATPase activity, though the latter declined over longer exposures, as in vivo . Silver flux over 3 h was small (<20%) relative to binding, and was insensitive to water chemistry . However, silver binding was decreased by elevations in freshwater Na(+) and dissolved organic carbon (humic acid) concentrations, increased by elevations in freshwater Cl(-) and reductions in pH, and insensitive to elevations in Ca(2+) . With the exception of the pH response, these effects were qualitatively and quantitatively similar to in vivo BLM responses . The results suggest that an in vitro BLM approach may provide a simple and cost-effective way for evaluating the protective effects of site-specific waters. Biochem Pharmacol, 2005 Jan 1, 69(1), 105 - 12 4-Hydroxynonenal inhibits cell proliferation and alters differentiation pathways in human fetal liver hematopoietic stem cells; Moneypenny CG et al.; During fetal development, the liver serves as the primary hematopoietic organ in which hematopoietic stem cells (HSC) comprise a large proportion of hepatic cell populations . Because HSC are capable of initiating long-term hematopoiesis, injury to these cells may have ramifications with regard to the etiology of blood-borne diseases . In the current study, we examined the effects of 4-hydroxynonenal (4-HNE), a mutagenic alpha,beta-unsaturated aldehyde that can be produced in utero, on HSC proliferation, differentiation, viability and apoptosis . Exposure of HSC to acute single doses of 4-HNE as low as 1nM inhibited HSC proliferation . Because 4-HNE rapidly disappears from culture media, a multiple dosing regime was also employed to approximate short-term steady state 4-HNE concentrations relevant to physiological oxidative stress . 4-Hydroxynonenal steady state concentrations as low as 1muM altered HSC differentiation pathways, but did not affect apoptosis or cause cell death . In contrast, exposure to steady state 5muM 4-HNE elicited a loss in viability, and increased the rate of apoptosis in total HSC populations . Collectively, our data indicate that cellular levels of 4-HNE associated with a low level of oxidative stress cause a loss of proliferation and viability and alter differentiation pathways in human fetal HSC. Lipids, 2004 Jul, 39(7), 649 - 58 Beta-oxidation, esterification, and secretion of radiolabeled fatty acids in cultivated Atlantic salmon skeletal muscle cells; Vegusdal A et al.; The white muscle of Atlantic salmon metabolizes FA with different chain lengths and different saturations at different rates, but few details are available on the processes involved or the products formed . We have investigated how multinucleated muscle cells (myotubes) in culture metabolize {1-(14)C}8:0, {1-(14)C}18:1n-9, and {1-(14)C}20:5n-3 . The myotubes were formed by the differentiation of isolated myosatellite cells from the white skeletal muscle of salmon fry . Almost all (98%) of the {1-(14)C}8:0 substrate was oxidized to acid-soluble products (ASP) and (14)CO2 after 48 h of incubation, whereas only approximately 50% of the {1-(14)C}18:1n-9 and {1-(14)C}20:5n-3 substrates were oxidized . However, only one cycle of beta-oxidation was measured by the method used . For all three substrates, the main ASP were acetate and a combined fraction of oxaloacetate and malate . Nearly twice as much radioactivity from the {1-(14)C}20:5n-3 substrate was found in the cellular lipids as radioactivity from {1-(14)C}18:1n-9, indicating that {1-(14)C}20:5n-3 was taken up into muscle cells more rapidly than {1-(14)C}18:1n-9 . Approximately 10% of the added {1-(14)C}20:5n-3 substrate and 5% of the added {1-(14)C}18:1n-9 substrate was secreted from the muscle cells into the culture media as esterified lipids . Immunocytochemical staining showed that the cells synthesized apolipoprotein A-I . Differentiated muscle cells also expressed peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta, two transcription factors that are involved in regulating beta-oxidation. Exp Parasitol, 2004 Nov-Dec, 108(3-4), 176 - 81 Trypanosoma cruzi: productive infection is not allowed by chorionic villous explant from normal human placenta in vitro; Lujan CD et al.; We hypothesize that a sustained infection of Trypanosoma cruzi into placental tissue might be diminished . Human placental chorionic villi and VERO cells as controls were co-cultured with T . cruzi . Parasites occupied 0.0137% at 3h, 0.0224% (24h), and 0.0572% (72 h) of the total chorionic villi area analyzed and some few placental samples were negative to parasite DNA, whereas 52% of VERO cells were infected at 3h and parasites occupied 0.57%, at 24h the parasite area was of 2.78% and at 72 h was of 3.32% . There were no live parasites in placenta-T . cruzi culture media at 72 h of co-culture . There were significantly increased dead parasites when T . cruzi was treated with unheated culture media coming from placental explants and fewer dead parasites when pre-heated culture media were employed . CONCLUSION: Low productive infection by T . cruzi into placental tissue associated with no viable parasites in the culture media partially due to placental thermo labile substances. J Hepatol, 2004 Dec, 41(6), 957 - 65 Stat3 confers resistance against hypoxia/reoxygenation-induced oxidative injury in hepatocytes through upregulation of Mn-SOD; Terui K et al.; BACKGROUND/AIMS: Hypoxia/reoxygenation (H/R) causes oxidative stress to the cell and induces apoptotic cell death . Signal transducer and activator of transcription-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and regeneration, and has recently been shown to protect cells against various pathogens . In order to investigate the hepatoprotective effects of Stat3, we examined whether it protects against H/R-induced injury in primary hepatocytes . METHODS: Primary cultured hepatocytes were prepared from SD rats . Adenoviruses and cytokines were added 2 days and 1h prior to the H/R insult, respectively . Hepatocytes and culture media were harvested for the assays before and after H/R insult . RESULTS: Interleukin-6 and cardiotropin-1, which may function mainly through Stat3 activation, protected cells from H/R-induced apoptosis . Adenoviral overexpression of the constitutively activated form of Stat3 (Stat3-C) reduced H/R-induced apoptosis as well as generation of reactive oxygen species (ROS) in hepatocytes . Interestingly, Stat3-C induced Mn-SOD, but not Cu/Zn-SOD, both at the protein and mRNA levels . Overexpression of Mn-SOD significantly reduced H/R-induced ROS and apoptosis by inhibiting redox-sensitive activation of caspase-3 activity . CONCLUSIONS: Stat3 protects hepatocytes from H/R-induced cell injury at least partly by upregulating Mn-SOD and inactivating caspase-3. J Biol Inorg Chem, 2004 Dec, 9(8), 954 - 60 Epub 2004 Dec. Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer's Abeta peptides; Huang X et al.; Nucleation-dependent protein aggregation ("seeding") and amyloid fibril-free formation of soluble SDS-resistant oligomers ("oligomerization") by hydrophobic interaction is an in vitro model thought to propagate beta-amyloid (Abeta) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimer's disease (AD), and other amyloid-associated neurodegenerative diseases . However, Abeta is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical Abeta deposition is abolished by genetic ablation of synaptic zinc in transgenic mice . We now present in vitro evidence that trace (<or=0.8 microM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic Abeta1-42-mediated seeding process and Abeta oligomerization . Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of Abeta1-40 (20 microM) initiated by Abeta1-42 (2 microM) were abolished by chelation of trace metal contaminants . Further, metal chelation attenuated formation of soluble Abeta oligomers from a cell-free culture medium . These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating Abeta amyloidosis and oligomerization. Endocrinology . 2004 Dec 2; {Epub ahead of print} Adrenomedullin is both proinflammatory and anti-inflammatory: its effects on gene expression and secretion of cytokines and macrophage migration inhibitory factor in NR8383 macrophage cell line; Wong LY et al.; Adrenomedullin (ADM) is a potent vasorelaxant peptide that plays important roles in cardiovascular homeostasis and inflammatory response . ADM derived from macrophages is one of the major sources of ADM which is produced in inflammatory process . To assess the functions of ADM in inflammation, we studied the temporal changes in ADM production and its effect on secretion of macrophage migration inhibitory factor (MIF) and cytokine response of NR8383 rat macrophages activated by lipopolysaccharide (LPS) . NR8383 cells were stimulated by LPS in the absence and presence of exogenous ADM, and the concentrations of ADM, MIF and proinflammatory cytokines (IL-6, TNF-alpha and IL-1 beta) in the culture media and gene expressions of the cells were measured . We confirmed that the secretion and mRNA expression of ADM in the macrophages were markedly increased by LPS . ADM increased initial secretion of MIF and IL-1 beta from both non-stimulated and LPS-stimulated cells, and it also increased basal and LPS-induced IL-6 secretion of the cells by 2 to 15-fold . However, it reduced secretion of TNF-alpha from LPS-stimulated cells by 34 to 56% . Our results suggest that ADM modulates MIF secretion and cytokine production and plays important roles in both the initiation and propagation of inflammatory response. Hum Reprod, 2005 Jan, 20(1), 61 - 71 Epub 2004 Dec 02. Morphological events in the primate endometrium in the presence of a preimplantation embryo, detected by the serum preimplantation factor bioassay; Rosario GX et al.; BACKGROUND: Hormonal modulation of the endometrium towards receptivity is well established; however, the role of embryonic stimuli in modulation of the endometrium prior to implantation, especially in primates, is unknown . The aim of the present study was to evaluate the endometrial histology when the embryo was present in its vicinity prior to implantation . METHODS: Preimplantation factor (PIF) bioassay was used as a tool to detect the presence of an embryo in the uterine lumen of mated bonnet monkeys (Macaca radiata) (n=9) . The control group comprised seven non-mated animals . The specificity of the PIF bioassay for the presence of an embryo was tested by studies in pregnant humans and monkeys . The effects of embryonic stimuli on the endometrial morphology were analysed by routine haematoxylin-eosin staining . The expressions of CD34, an endothelial cell marker, alpha-smooth muscle actin (alpha-SMA), a marker for blood vessel maturation, and prolactin, a marker of endometrial decidualization, were studied by immunohistochemistry . RESULTS: That PIF is embryo specific was established by its presence in sera of pregnant humans, monkeys and also in embryo culture media . Six mated bonnet monkeys were found to be PIF positive . Morphologically, the endometria from these PIF-positive animals showed the presence of the pre-epithelial plaque reaction, increased angiogenesis and stromal compaction . The significantly increased number of CD34- and alpha-SMA-positive blood vessels (P<0.05) in the endometria of PIF-positive animals indicated increased angiogenesis in response to embryonic stimuli . The endometrial expression of immunoreactive prolactin was also significantly increased (P<0.05) in the PIF-positive animals, indicating decidualization . CONCLUSIONS: Using PIF as a marker to detect early pregnancy in bonnet monkeys, we have shown that the embryo induces a pre-epithelial plaque type of reaction, increased angiogenesis and decidual reaction in the endometrium prior to implantation. Appl Environ Microbiol, 2004 Dec, 70(12), 7295 - 302 Detection and quantification of Wallemia sebi in aerosols by real-time PCR, conventional PCR, and cultivation; Zeng QY et al.; Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease . The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi . Thus, its occurrence in different environments has often been underestimated . In this study, we developed two sets of PCR primers specific to W . sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples . Both PCR systems proved to be highly specific and sensitive for W . sebi detection even in a high background of other fungal DNAs . These methods were employed to investigate the presence of W . sebi in the aerosols of a farm . The results revealed a high concentration of W . sebi spores, 10(7) m(-3) by real-time PCR and 10(6) m(-3) by cultivation, which indicates the prevalence of W . sebi in farms handling hay and grain and in cow barns . The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W . sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment. Sichuan Da Xue Xue Bao Yi Xue Ban, 2004 Nov, 35(6), 806 - 8 {Effects of leptin on development of mouse preimplantation embryos in vitro}; Chen Q et al.; OBJECTIVE: To investigate the effects of leptin on development of mouse preimplantation embryos in vitro . METHODS: (1) Groups of 2-cell stage embryos randomly selected were placed in drops of CZB medium with or without recombinant leptin (10, 50, 100 and 500 ng/ml) and were cultured to the hatched blastocyst stage, and then embryo-transfer was carried out and the implantation rate was observed and determined . (2) Groups of 2-cell stage embryos randomly selected were placed in drops of leptin free CZB medium and cultured to morula stage . Then we changed the medium, randomized the embryos into CZB medium with or without recombinant leptin (10, 50, 100 and 500 ng/ml), and cultured them to the hatched blastocyst stage . RESULTS: Addition of leptin to embryo culture media promoted the development from 2-cell stage embryo to morula, blastocyst and hatched blastocyst and improved the implantation rate . Leptin at 0, 10, 50 ng/ml concentrations caused a dose-dependent promotion, nevertheless Leptin at even higher concentrations (100 and 500 ng/ml) only brought on the same promotion as what the Leptin at concentration of 50 ng/ml did (P>0.05) . Addition of leptin to embryo culture media seemed to have no effects on the in vitro development from morula to blastocyst and hatched blastocyst . CONCLUSION: Leptin plays an important role in the development of preimplantation embryo . It can promote embryo development and embryo implantation. Indian J Exp Biol, 2004 Aug, 42(8), 758 - 65 Role of L-lysine HCl in adoptive immune therapy towards development of suitable tuberculosis vaccination; Dasgupta S et al.; L-Lysine HCI is being proposed to be a possible biocompatible adjuvant to enhance immune response by virtue of its probable non-specific bridging action and cellular proliferation properties . This proposal has been tried to be substantiated by designing an in vitro culture protocol, varying the concentration of L-lysine HCI and its further in vivo application . Splenic lymphocyte population has been extracted from mice and co-cultured with extracted mice macrophage population in presence of either Bacille Calmette Guerrin (BCG) or Hepatitis B surface antigen (HbsAg) and added L-lysine hydrochloride in culture media . Post incubation of these cultures, "taught" cell population has been adoptively transferred in naive mice . These mice were then challenged by respective antigen dose, Change in Immune response with this challenge was noted . Antibody titre was followed in all the experiments as a measure of immune response . In adoptive immune transfer experiment of with HbsAg (AIT-HbsAg), similar to that with BCG (AIT-BCG), after the incubation period, antibody titre was higher in added lysine containing cultures in comparison with the control ones . Post transfer followed by antigen challenge, in AIT-BCG the expected augmentation in immune response was hardly visible . But in AIT-HbsAg, with the help of lysine booster, the animals responded better as far as the antibody titre is concerned. Zentralbl Gynakol, 2004 Dec, 126(6), 368 - 72 {Optimisation of possible success in an IVF program}; Ludwig M et al.; The success of an IVF (in vitro fertilisation) programme is mainly dependent an the quality of the IVF laboratory . Beside this the quality of ovarian stimulation play another, but not as important role . In the 4 (th) quarter of 2003 we have changed the culture media for oocytes and embryos in the IVF laboratory of ENDOKRINOLOGIKUM Hamburg from simple to complex media (G-1 version 3) . As protein source we used HSA-solution . Before embryo transfer equilibration for 1 hour in EmbryoGlue was performed, which contains hyaluran . Hyaluran is said to improve implantation rates by different ways . The media were bought from Vitrolife Schweden AB (Kungsbacka, Schweden) via a German distributor . These data were compared to those of two previous 3-month-periods (3rd quarter 2003 und 4th quarter 2002) . The study design was prospective, with two historical control groups . In the three study periods 255, 343 and 503 patients were treated by either conventional IVF or ICSI (intracytoplasmic sperm injection) . The cohorts were comparable regarding their anamnestic data . The number of patients treated which the GnRH antagonist cetrorelix (Cetrotide) significantly increased from 24.9 % to 49.5 % and 60.9 % . Under this protocol significantly less oocytes were retrieved (10.23 vs . 8.73 vs . 8.88), leading to slightly less embryos (1.94 vs . 1.91 vs . 1.85, not significant, n . s.) . The cumulative embryo score significantly decreased from 28.58 to 24.10 . It was significantly higher in the study cohort as compared to the two control cohorts (32.44) . The clinical pregnancy rate significantly increased to 25.29 % in the study cohort as compared to the two historical control cohorts (15.02 % and 18.75 %) . We could achieve a significant improvement in the success rates of our IVF program by switch from simple to more complex culture media in the IVF laboratory . The use of the GnRH antagonist protocol with cetrorelix (Cetrotide) lead to more comfort to the patients but did not influence the results. Zhonghua Yu Fang Yi Xue Za Zhi, 2004 Nov, 38(6), 415 - 8 {Effects of vitamin D analogue EB1089 on proliferation and apoptosis of hepatic carcinoma cells.}; Luo WJ et al.; OBJECTIVE: This study aimed at investigating the effects of vitamin D analogue EB1089 on the proliferation and apoptosis of hepatic carcinoma cells . METHODS: Hepatic carcinoma cell strain G(2) (Hep-G(2)) in which prominent vitamin D receptor (VDR) mRNA could be expressed and the cell strain T (HCC-T) negative in VDR gene expression were incubated in culture media with 100 nmol/L, 10 nmol/L and 1 nmol/L EB1089 for 2 d, 4 d and 6 d, respectively . Survival and proliferation of the cells were detected by blue tetrazolium colorimetric test and plate clone-forming test, the VDR mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and apoptosis of the cells was detected by flow cytometry (FCM) and electron microscopy . RESULTS: EB1089 could inhibit the proliferation of hepatocellular cell line Hep-G(2) that expressed prominent vitamin D receptor mRNA, the inhibitory rate is 17.5% approximately 72.1% . On the other hand, EB1089 had no anti-proliferative effect on hepatocellular cell line HCC-T in which the gene expression of vitamin D receptors was negative . The electron microscope results showed that EB1089 could induce apoptosis of hepatocarcinoma cells and the percentages of apoptotic cells measured by flow cytometer was 21.4% . Cell cycle progression was blocked at G(1) phase with EB1089 . CONCLUSION: EB1089 could inhibit proliferation of human Hep-G(2), probably through VDR, and induce apoptosis of the cells. Kidney Int, 2004 Dec, 66(6), 2329 - 36 Lead exposure raises superoxide and hydrogen peroxide in human endothelial and vascular smooth muscle cells; Ni Z et al.; BACKGROUND: Chronic lead exposure causes hypertension and cardiovascular disease, which are associated with, and, in part, due to oxidative stress . While occurrence of oxidative stress in lead-exposed animals and cultured endothelial cells has been well-established, direct and specific evidence on the type of the reactive oxygen species (ROS) produced by lead-exposed vascular cells is lacking and was investigated . METHODS: Human coronary endothelial (EC) and vascular smooth muscle cells (VSMC) were incubated in appropriate culture media in the presence of either 1 ppm or 10 ppm lead acetate or sodium acetate (control) for 1 to 30 minutes or 60 hours . Productions of superoxide and hydrogen peroxide in the cell populations were determined by flow cytometry using hydroethidine and dihydrorhodamine, respectively . Data from a minimum of 10,000 cells were collected and analyzed using Cell Quest software . In addition, Cu Zn superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and NAD(P)H oxidase (gp91phox) were measured . RESULTS: Short-term lead exposure resulted in a significant rise in both superoxide and hydrogen peroxide production by both EC and VSMC . After long-term exposure, detectable superoxide levels fell to near normal level, while hydrogen peroxide production remained high . This was associated with up-regulations of gp91phox, elevation of superoxide dismutase, reduction of VSMC catalase, and no change in GPX levels . Together, these events can account for the observed decline in superoxide and the rise in hydrogen peroxide following long-term lead exposure . CONCLUSION: Lead exposure promotes generation of superoxide and hydrogen peroxide in human EC and VSMC . This phenomenon can potentially contribute to the pathogenesis of the lead-associated hypertension and cardiovascular disease, and points to the potential benefit of lowering lead burden in the exposed populations. J Nat Prod, 2004 Nov, 67(11), 1796 - 9 Anti-inflammatory phloroglucinols and terpenoids from Garcinia subelliptica; Weng JR et al.; Three new phloroglucinols, garcinielliptones K (1), L (2), and M (3), and two new terpenoids, garcinielliptones N (4) and O (5), have been isolated from the seeds of Garcinia subelliptica . The structures of 1-5 including their relative configurations were elucidated by spectroscopic methods and supported by computer-generated molecular modeling . Compounds 2 and 3 showed potent inhibitory effects on the release of beta-glucuronidase, and on beta-glucuronidase and histamine, respectively, from peritoneal mast cells stimulated with p-methoxy-N-methylphenethylamine (compound 48/80) in a concentration-dependent manner . Compounds 2 and 3 showed potent effects on NO production in culture media of RAW 264.7 cells in response to lipopolysaccharide (LPS) . Compound 2 also showed a potent effect on NO production in culture media of N9 cells in response to LPS/interferon-gamma (IFN-gamma). J Assist Reprod Genet, 2004 Aug, 21(8), 291 - 5 Prospective randomized comparison of two embryo culture systems: P1 medium by Irvine Scientific and the Cook IVF Medium; Ben-Yosef D et al.; PURPOSE: To compare the efficacy of two commercially available in vitro fertilization (IVF) and embryo culture media systems: the glucose-free P1 Medium supplemented with 20% synthetic serum substitute (SSS) (Irvine Scientific), and the Cook IVF Medium (Cook, Australia) . METHODS: A prospective randomized study . Medical center-based IVF Unit affiliated to the Faculty of Medicine of Tel Aviv University . IVF patients were randomly assigned to either P1 Medium supplemented with 20% SSS (182 patients, 196 cycles) or Cook Medium (167 patients, 179 cycles) . RESULTS: Fertilization rates were similar with both media (52.3 +/- 26.1 and 53.8 +/- 27.6, respectively) . Likewise, no difference was found in morphological characteristics and grading of cultured embryos . However, a significantly higher proportion of the embryos incubated in the P1 Medium reached the four-cell stage on day 2 or the 6-cell stage on day 3 postfertilization, compared to those incubated in Cook Medium (54.3% vs . 41.9%, p < 0.0001) . Clinical pregnancy and delivery rates were improved when oocytes and embryos were cultured in P1 Medium . Finally, Implantation rate was significantly higher in the P1 Medium Group (9.9% vs . 6%, respectively) . CONCLUSIONS: Our results suggest that the P1 Medium may be associated with a higher embryo cleavage rate and improved implantation rates compared to the Cook IVF Medium. Di Yi Jun Yi Da Xue Xue Bao, 2004 Nov, 24(11), 1248 - 50 {Biological effect of serum of kidney-tonifying traditional Chinese drug-fed rats on cultured osteoblasts.}; Tang JG et al.; OBJECTIVE: To investigate the biological effects of the serum of rats fed with kidney-tonifying traditional Chinese drugs on cultured osteoblasts in vitro . METHODS: The culture media containing the serum from rats fed with the drugs at different doses (high, mediate and low doses) were used to treat the second passage of the osteoblasts from the skull of newborn SD rats, and the cell proliferation, differentiation and mineralization were observed . RESULTS: The serum stimulated the cell proliferation, enhanced alkaline phosphatase activity and increased the number of mineralized nodules in the cultured osteoblasts . CONCLUSION: Kidney-tonifying traditional Chinese drugs can stimulate the proliferation, differentiation and maturation of cultured osteoblasts in vitro. Hepatobiliary Pancreat Dis Int, 2004 Nov, 3(4), 543 - 7 Effects of cell cycle on telomerase activity and on hepatitis B virus replication in HepG2 2.2.15 cells; Huang YQ et al.; BACKGROUND: It has been shown that telomerase activity and hepatitis B virus (HBV) replication are closely associated with cell cycle . This study aimed to further investigate the effects of cell cycle on telomerase activity and on HBV replication . METHODS: Human hepatoma cells transfected with HBV DNA (HepG2 2.2.15 cell line) were treated respectively with serum deprivation, all-trans retinoic acid (RA), dimethyl sulfoxide (DMSO), or sodium butyrate . The cell cycle of HepG2 2.2.15 cells was analyzed by flow cytometry . The telomerase activities of the cells were detected by TRAP-PCR-ELISA . HBV DNA in culture medium was assayed by a fluorescent quantitative PCR assay and a semiquantitative dot blot hybridization technique . HBsAg and HBeAg in culture media were quantitatively examined by an ELISA assay . RESULTS: Treatments with serum deprivation, RA, DMSO, or sodium butyrate inhibited the proliferation of HepG2 2.2.15 cells and led to cell arrest in the G0/G1 phase of cell cycle . The percentage of the G0/G1 phase in the groups of sodium butyrate, DMSO, RA and serum-free was 85.2%, 71.9%, 68.3% and 65.2%, respectively, but in the control group, 43.1% (P<0.01) . The activities of telomerase of the cells were also significantly inhibited by 82.8%, 74.6%, 76.1% and 69.4% respectively . In addition, HBV replication of the HepG2 2.2.15 cells remarkably increased as shown by the contents of HBV DNA, HBsAg and HBeAg in the culture media of the cells treated with sodium butyrate, DMSO, RA or serum deprivation (P<0.01) . The amounts of HBV DNA in the groups of sodium butyrate, DMSO, RA, serum deprivation and control were 6.7X10(6), 4.8X10(6), 4.4X10(6), 5.1X10(6) and 1.2X10(6) copies/ml, respectively (P<0.01) . Telomerase was expressed mainly in the cells in S phase . HBV replication increased in quiescent cells (G0/G1 phase), and decreased in proliferating phase (S phase) . CONCLUSION: The current data approve that HBV replication is associated with the cellular proliferative activity. Cell Biol Int, 2004, 28(12), 863 - 73 Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro; Zhang C et al.; Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-beta (TGF-beta) in various renal diseases . To elucidate the role of CTGF in renal tubular epithelial-myofibroblast transdifferentiation, we examined the expression of alpha-smooth muscle actin (alpha-SMA), vimentin, tenascin-C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF-beta . It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 microg/L), the expression of alpha-SMA and tenascin-C mRNA increased significantly (p<0.01), while collagen IV gene expression decreased significantly (p<0.01), all in a dose-dependent manner . The percentage of alpha-SMA-positive cells was significantly larger in the rhCTGF-stimulated groups than that in negative control (38.9%, 65.5% vs . 2.4%, respectively, p<0.01) as confirmed by flow cytometry . Both cytoplasmic and secretory tenascin-C expression was upregulated by the stimulation of rhCTGF (p<0.01) . Under this condition, collagen IV secreted into the culture media was lowered markedly (p<0.01) . On RT-PCR analysis, TGF-beta1 upregulated CTGF gene expression, preceding that of alpha-SMA . The alpha-SMA mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p<0.01) . With prolonged incubation time, CTGF antisense ODN also inhibited intracellular alpha-SMA protein synthesis, as demonstrated by indirect immuno-fluorescence . So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis. Laryngoscope, 2004 Dec, 114(12), 2161 - 7 Therapeutic potential of growth factors for aging voice; Hirano S et al.; OBJECTIVES/HYPOTHESIS: It has been reported that in aged vocal folds, dense collagen deposition takes place and hyaluronic acid decreases in the lamina propria, which are thought to contribute to the vocal problems occurring with age (presbyphonia) . To restore aged vocal folds to their younger state, it seems crucial to address these age-related lamina propria changes . Intervention that might increase hyaluronic acid and decrease collagen would appear to be a potentially useful approach . The present study examined the effects of growth factors on aged fibroblasts in terms of the production of hyaluronic acid and collagen type I . STUDY DESIGN: In vitro study using animal model . METHODS: Fibroblasts were harvested from young and aged rat vocal folds and cultured with or without hepatocyte growth factor and/or basic fibroblast growth factor at different concentrations . Subsequently, the production of hyaluronic acid and collagen type I was examined in the supernatant culture media using enzyme-linked immunosorbent assay . RESULTS: Aged fibroblasts produced less hyaluronic acid than younger fibroblasts . When aged and young fibroblasts were cultured with basic fibroblast growth factor, hyaluronic acid production increased and collagen type I production decreased regardless of the concentration, whereas the effects of hepatocyte growth factor appeared to vary with concentration . The basic fibroblast growth factor also was associated with stimulation of growth of aged fibroblasts . CONCLUSION: The present results suggest that growth factors, especially basic fibroblast growth factor, may have therapeutic potential in restoration of aged vocal fold. Circ J, 2004 Dec, 68(12), 1230 - 2 Production of endothelin-1 and big endothelin-1 by human cardiac myxoma cells--implications of the origin of myxomas--; Sakamoto H et al.; BACKGROUND: Although the origin of cardiac myxomas is still controversial, the 2 main hypotheses are that the tumor cells originate either from multipotential mesenchymal cells or from endocardial neural tissue . METHODS AND RESULTS: The production of various cytokines in 2 human cardiac myxoma cell lines was examined by enzyme-linked immunosorbent assay . After 7 days of culture, extremely high concentrations of interleukin-6 were detected in the culture media from both myxoma cell lines . Increased production of CXC chemokines, interleukin-8 and growth-related oncogene-alpha, were observed in both myxoma cell lines . Endothelin (ET)-1 and its precursor, big ET-1, were detected in the culture media from both myxoma cell lines . The production of both ET-1 and big ET-1 by myxoma cells was higher than by human umbilical vein endothelial cells . Similar to endothelial cells, myxoma cells did not produce stem cell factor, granulocyte colony-stimulating factor, hepatocyte growth factor, or ET-3 . CONCLUSIONS: The similarity of the cytokine production pattern between cardiac myxoma cells and endothelial cells supports the hypothesis that the tumor cells originate from mesenchymal cells capable of endothelial differentiation . Overproduction of CXC chemokines may explain, in part, the malignant potential of histologically benign myxomas. J Neurosci, 2004 Nov 24, 24(47), 10642 - 51 Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology; Shihabuddin LS et al.; Niemann-Pick disease is caused by a genetic deficiency in acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes . In the present study, we evaluated the effects of direct intracerebral transplantation of neural progenitor cells (NPCs) on the brain storage pathology in the ASM knock-out (ASMKO) mouse model of Type A Niemann-Pick disease . NPCs derived from adult mouse brain were genetically modified to express human ASM (hASM) and were transplanted into multiple regions of the ASMKO mouse brain . Transplanted NPCs survived, migrated, and showed region-specific differentiation in the host brain up to 10 weeks after transplantation (the longest time point examined) . In vitro, gene-modified NPCs expressed up to 10 times more and released five times more ASM activity into the culture media compared with nontransduced NPCs . In vivo, transplanted cells expressed hASM at levels that were barely detectable by immunostaining but were sufficient for uptake and cross-correction of host cells, leading to reversal of distended lysosomal pathology and regional clearance of sphingomyelin and cholesterol storage . Within the host brain, the area of correction closely overlapped with the distribution of the hASM-modified NPCs . No correction of pathology occurred in brain regions that received transplants of nontransduced NPCs . These results indicate that the presence of transduced NPCs releasing low levels of hASM within the ASMKO mouse brain is necessary and sufficient to reverse lysosomal storage pathology . Potentially, NPCs may serve as a useful gene transfer vehicle for the treatment of CNS pathology in other lysosomal storage diseases and neurodegenerative disorders. Hua Xi Kou Qiang Yi Xue Za Zhi, 2004 Oct, 22(5), 366 - 9 {Insulin-like growth factor-II and basic fibroblast growth factor affect periodontal ligament cells expressing osteoprotegerin in vitro}; Ling JQ et al.; OBJECTIVE: This study was carried out to investigate the effects of insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) on osteoprotegerin (OPG) secretion of periodontal ligament cells (PDLCs) . METHODS: Healthy human premolars extracted for orthodontic reasons from 12-14 years old donators were obtained, and periodontal tissues were collected and cultured to obtain PDL cells . Primary or first passage PDLCs were cloned by means of limited dilutions . PDLCs with osteoblastic phenotypes were characterized as follows: Alkaline phosphatase activity, collagen III production and bone-like nodules formation . IGF-II and bFGF were added into culture media and their effects on PDLCs proliferation and OPG secretion were observed . The OPG concentrations in cell culture supernatants were detected by sandwich ELISA . Living cell numbers were demonstrated by MTT test . The average levels of OPG secretion by a single cell were calculated by dividing OPG concentration with MTT-test result . RESULTS: Both IGF-II and bFGF upregulated the mtt values (P < 0.05), but ICF-II downregulated the opg/mtt values (P < 0.05), whereas bFGF had no significant effect on opg/mtt values (P > 0.05) . CONCLUSION: IGF-II enhances the proliferation of PDL cells but prohibits OPG secretion . Although bFGF has the same effect on the proliferation of PDL cells, it has no effect on OPG secretion . Before cytokines were used to enhance periodontal regeneration, their effects on local bone balance should also be studied. Commun Agric Appl Biol Sci, 2004, 69(1), 15 - 22 The hemolymph of caterpillars Spodoptera littoralis: physico-chemical properties and ionic composition compared to culture media; Smagghe G et al.; In this paper, we determined some physico-chemical properties like osmotic pressure, pH and electrical conductivity of the hemolymph from caterpillars of Spodoptera littoralis (Lepidoptera: Noctuidae) during the last larval instar . It was of interest that we observed an increase in osmotic pressure with the increase in age in the last instar that may concur with the start of histolysis at metamorphosis . These physicochemical properties were then compared to those of Grace's and modified Grace's tissue culture medium . In addition, concentrations of the cations Na, K, Ca and Mg, and the anions Cl, NO3, PO4 and SO4 were determined in the insect hemolymph of S . littoralis . The cations K and Mg reached high values with a percent of about 52% of the total amount of cations . The concentration of sodium was low . The total sum of the anions consisted about 56 meq/1, and this allows to neutralise about 45 % of the total cations. Gac Med Mex, 2004 Sep-Oct, 140(5), 507 - 12 {Isolation and characterization of wild Sporothrix schenkii strains and investigation of sporototrichin reactors}; Sanchez-Aleman MA et al.; We conducted a study in the southern mountains of the Mexican State of Oaxaca that consisted of isolation of wild Sporothrix schenckii strains obtained from soil samples and investigation of positive reactors to skin test reaction with sprotrichin antigen . The study was conducted by means of recollection of soil samples and processing of these with dilution methods and fungal isolation in ordinary culture media Sabouraud simple Agar with and without antibiotics (SS, SA) . Suspected strains underwent dimorphism, melanin formation, and virulence confirmation tests . Investigation of positive reactors to sporotrichin Y (yeast) was also conducted . Three supposed strains were identified due to their reproductive characteristics, melanin production, and virulence . In the community, 144 individuals were studied, of whom 6.25% were positive to sporotichin . Isolation of virulent strains of Sporothrix schenkii from nature (soil) and primoinfection of a percentage of the studied population were confirmed. Int J Dev Biol, 2004, 48(8-9), 771 - 82 A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions; Sullivan CH et al.; Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation . We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds . We first examined the issue of specification of head ectoderm for a lens fate . A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm . This positive lens response was observed in cultures grown in a wide range of culture media . We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response . Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme . This mesenchyme is infiltrated by neural crest cells in most regions of the head . Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area. Invest Ophthalmol Vis Sci, 2004 Dec, 45(12), 4302 - 11 Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells; Li DQ et al.; PURPOSE: To investigate whether exposure of human corneal epithelial cells to hyperosmotic stress activates the c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway, and stimulates production of the matrix metalloproteinases (MMPs): gelatinase (MMP-9), collagenases (MMP-1 and -13), and stromelysin (MMP-3) . METHODS: Primary human corneal epithelial cells cultured in normal osmolar medium (312 mOsM) were exposed to media with higher osmolarity (350-500 mOsM) achieved by adding NaCl, with or without SB202190, an inhibitor of the JNK pathway; dexamethasone; or doxycycline for different lengths of time . The conditioned media were collected after 24 hours of exposure for zymography and ELISA . Total RNA was extracted from cultures treated for 6 hours and subjected to semiquantitative RT-PCR . Cells treated for 5 to 60 minutes were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-c-Jun . JNK1 activation was also detected with an immunoassay system . RESULTS: The concentrations of MMP-9, -1, and -3 proteins in 24-hour conditioned media of corneal epithelial cells progressively increased as the media's osmolarity was increased from 312 to 500 mOsM by the addition of NaCl . The concentration of MMP-13 progressively increased to a peak at 450 mOsM . Active p-JNK-1, p-JNK-2, and p-c-Jun were detected by Western blot as early as 5 minutes and peaked at 60 minutes in cells exposed to hyperosmolar media . The levels of p-JNK-1, p-JNK-2, and p-c-Jun correlated positively with the osmolarity of the culture media . The p-JNK inhibitor SB202190 and doxycycline markedly inhibited the stimulation of p-JNK-1, p-JNK-2, and p-c-Jun, as well as MMP-9, -1, -13, and -3 at both the mRNA and protein levels in the cells exposed to hyperosmolar media . CONCLUSIONS: Expression and production of MMP-9, -1, -13, and -3 by human corneal epithelial cells correlated positively with increasing media osmolarity . This increase was mediated at least in part through activation of the JNK SAPK pathway . Doxycycline, an agent used to treat MMP-mediated ocular surface disease, inhibited the hyperosmolarity-induced MMP production and JNK activation . The relevance of these findings to stimulated production of MMPs by the elevated tear osmolarity in dry eye remains to be determined. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Oct, 21(5), 727 - 31 {The influence of Bazhen decoction on hematopoietic modulator in anaemic mice}; Chun Z et al.; This study was designed to evaluate the effect of Bazhen decoction on bone marrow depression induced by cyclophosphamide (CY) in mice . An experimental model of mouse bone marrow injury was established through cyclophosphamide induced and the following phenomena were observed . The techniques of culture of hematopoietic progenitor cell and hematopoietic growth factor assay were used . Bazhen decoction could obviously promote the proliferation of bone marrow cells of anaemic mice . The culture media of spleen cell, macrophage, lung and skeletal muscle treated with Bazhen decoction had much stronger stimulating effects on hematopoietic cells . The bone marrow cells of the anaemic mice could yield TNF through Bazhen decoction treatment . It was suggested that Bazhen decoction is clinically a hopeful drug used to cure bone marrow depression and attenuate the side effects of CY. Prikl Biokhim Mikrobiol, 2004 Sep-Oct, 40(5), 571 - 8 {Extracellular polymers in callus cultures of Fagopyrum tataricum (L.) Gaertn . with different morphogenic activities: time courses during the culture cycle}; Cycloheximide prevents production of arresting et al.; Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia, Lima, Peru . iiad@upch.edu.peAIM: To evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermatogenesis and sperm count in male rats . METHODS: The study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections . The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined . RESULTS: The fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly . Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls . The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control . CONCLUSION: The difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules. Endocrinology, 2005 Feb, 146(2), 702 - 12 Epub 2004 Nov 11. Miniglucagon (MG)-Generating Endopeptidase, which Processes Glucagon into MG, Is Composed of N-Arginine Dibasic Convertase and Aminopeptidase B; Fontes G et al.; Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg(17)-Arg(18) dibasic site, displays biological effects opposite to that of the mother-hormone . This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon . We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase . We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG . Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium . We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH(4)C(1)) . Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans . By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG) . Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia . We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations. Rev Iberoam Micol, 2004 Jun, 21(2), 96 - 9 {Growth in species of the genus Ascobolus . II . (Pezizales-Ascomycota)}; Dokmetzian DA et al.; The kinetics of growth of six heterothallic species of the genus Ascobolus was studied in liquid culture media . The results obtained showed variation among the species in the duration of the different phases of the growth cycle . Four groups can be recognized considering the extension of the exponential phase of growth . The stationary phase, which differs in its length, is frequently very short, entering quickly in the phase of death, accompanied by the autolysis of the mycelium. Scanning, 2004 Sep-Oct, 26(5), 209 - 16 Atomic force microscopy imaging of retroviruses: human immunodeficiency virus and murine leukemia virus; Kuznetsov YG et al.; Retroviruses are membrane-enveloped, RNA-containing viruses that produce a wide range of threatening diseases in higher animals . Among these are human immunodeficiency virus (HIV), which produces acquired immune deficiency syndrome (AIDS) in humans, and murine leukemia virus (MuLV), which produces leukemias in rodents . We have obtained the first atomic force microscopy (AFM) images of these two retroviruses, both isolated from culture media and emerging from infected cell surfaces . The HIV virions are 127 nm diameter on average, and those of MuLV are 145 nm, although there are wide distributions about the means . The AFM images show the arrangement of the envelope protein, responsible for host cell entry, on the surfaces of both virions . Disruption of the viruses using detergents or physical means allowed us to visualize interior structures, including the outer shells of both MuLV and HIV, the cores of MuLV, and the nucleic acid of HIV complexed with core proteins . Using immunolabeling techniques borrowed from electron microscopy, we were able to demonstrate the binding of gold-labeled antibodies directed against the envelope protein of MuLV . The AFM images are revealing, not only in terms of surface topology, but in terms of interior features as well, and they reveal the eccentricities and uniqueness of individual virus particles rather than yielding the average member of the population . Further application of AFM to viruses associated with other pathologies may ultimately have a significant impact on the diagnosis and treatment of virus-promoted diseases. J Med Entomol, 2000 May, 37(3), 316 - 8 Oviposition and maintenance of Forcipomyia (Lasiohelea) townsvillensis (Diptera: Ceratopogonidae) in the laboratory; Cribb BW; Fecundity, oogenesis, oviposition, and percentage egg hatch were quantified for the blood-feeding midge Forcipomyia (Lasiohelea) townsvillensis (Taylor) . Data are similar to that reported for other species of blood-feeding Forcipomyia . Eggs rarely developed from a partial blood meal but invariably developed after a single, complete blood meal . Results suggest that this species is anautogenous . Oviposition media were investigated and a successful medium and holding chamber type identified . Longevity of adults in the laboratory was studied and indicates the possibility for >1 gonotrophic cycle to occur . Adult survival at different relative humidities showed midges can survive 35-98% RH . Rearing of larvae in the laboratory and culture media are discussed . The data supplied in this paper provide the basis for the laboratory culture of F . (L.) townsvillensis. J Cell Physiol . 2004 Nov 8; {Epub ahead of print} Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells; Yan Q et al.; The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation . To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro . We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture . However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells . Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells . Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media . The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells . A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus . SPARC does not appear to have a strong nuclear localization sequence . Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion . SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis . (c) 2004 Wiley-Liss, Inc. Comp Biochem Physiol C Toxicol Pharmacol, 2004 Jul, 138(3), 251 - 8 Developmental expression of aquaporin-3 in zebrafish embryos (Danio rerio); Lance SL et al.; Fish embryos have never been successfully cryopreserved because of the low permeability of cryoprotectants into the yolk . Recently, we used aquaporin-3 fused with a green fluorescent protein (AQP3GFP) to modify the zebrafish embryo, and demonstrated that the pores functioned physiologically . This increased the water and cryoprotectant permeability of the membranes . We have continued our work on AQP3-modified embryos and here we report their developmental expression of AQP3, the success of various culture media on their survival and development, and their reproductive success . The AQP3GFP expression begins within 30 m after the mRNA AQP3GFP injection into the yolk of the 1- to 4-cell embryo . This expression is distributed in the membranes throughout the blastoderm and the yolk syncytial layer within 24 h . It diminishes after 96 h . We found no difference in the survival or normal development of embryos from AQP3GFP or wild-type adults . Additionally, zebrafish embryos did not require special culture medium to survive after AQP3GFP modification . In fact, they survived best in embryo medium (ca . 40 mOsm) . Embryos reared entirely in embryo medium had a higher percent survival and a higher percent normal development than those exposed to a high osmolality sucrose culture medium (ca . 330 mOsm) . The mechanism whereby these embryos can maintain their internal osmolality in a hypoosmotic solution with water channels in their membranes is unknown. Virology, 2004 Dec 5, 330(1), 168 - 77 Differential ability of two simian virus 40 strains to induce malignancies in weanling hamsters; Vilchez RA et al.; Different strains of simian virus 40 (SV40) exist and are associated with some human malignancies, but it is not known if SV40 strains differ in biological potential in vivo . In two long-term experiments, Syrian golden hamsters 21 days of age were inoculated by the intraperitoneal route with two different strains of SV40 (10(7) plaque-forming units/animal) and were followed for 8 or 12 months . In vivo responses to strain VA45-54, isolated originally from monkey kidney cells, and to strain SVCPC, recovered from human cancers, were compared . Control animals of the same age were inoculated intraperitoneally with cell culture media . Malignancies developed only in animals infected with SV40 and not in controls . The rate of tumor development was more frequent among animals infected with strain SVCPC than with VA45-54, both in experiments held for 8 months (11/22, 50% vs . 4/20, 20%) and for 12 months (7/15, 47% vs . 3/13, 23%) . Histologically, the tumors resembled mesotheliomas, osteosarcoma, and poorly differentiated sarcomas . Metastases to lung and lymph nodes occurred with both viral strains . T-antigen expression was detected in most tumor cells by immunohistochemistry . Anti-T-antigen antibodies were produced by almost all tumor-bearing animals and by about two-thirds of those that did not develop tumors after virus inoculation . SV40 viral neutralizing antibodies were detected in all tumor-bearing animals and in 92% and 38% of those inoculated with SVCPC and VA45-54, respectively, that failed to develop tumors . Antibody titers were usually higher in animals with tumors than in those without . Control animals did not develop viral antibodies . Infectious virus was recovered from 2 of 15 tumors tested . This study showed that there are biological differences between these two SV40 strains that influence the outcome of infections in normal hosts, including the development of malignancies and neutralizing antibody, and proved the principle that SV40 strains from different clades can vary in biological properties in vivo. J Cell Biochem, 2005 Jan 1, 94(1), 139 - 52 Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3; Teillaud C et al.; The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-{MARRS}bp, with a molecular weight of 65 kDa, in several tissues and species . Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues . In order to investigate the expression of 1,25D3-{MARRS}bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-{MARRS}bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3) . Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-{MARRS}bp in MO6-G3 cells . Moreover, 1,25D3-{MARRS}bp was up-regulated, in vivo, in differentiated dental cells . Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence . Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-{MARRS}bp expression with a maximal effect at 50 nM steroid . In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-{MARRS}bp . Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells . Altogether, these results indicate that the 1,25D3-{MARRS}bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3 . In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells . 2004 Wiley-Liss, Inc. Zygote, 2004 Aug, 12(3), 263 - 7 Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes; Yi YJ et al.; This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes . The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively . Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively . Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate . Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations . A similar pattern was observed for polyspermic penetration and male pronucleus formation . The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations . The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations . The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air. Transplant Proc, 2004 Sep, 36(7), 1980 - 4 Effect of pirfenidone on induction of chemokines in rat hepatocytes; Kaibori M et al.; BACKGROUND: Hepatic ischemia-reperfusion results in a neutrophil-dependent liver injury . The process of neutrophil recruitment and activation in this injury is at least partially dependent on the induction of chemokines, such as cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) in rats . In the liver, parenchymal cells (hepatocytes), in addition to nonparenchymal cells such as Kupffer cells, have been reported to produce chemokines in the regulation of hepatic inflammation . Pirfenidone (PFD) is a new experimental drug used as an antifibrotic agent . Studies were performed to determine whether PFD influences the production of CINC and MIP-2 stimulated by interleukin (IL)-1beta in a primary culture model of rat hepatocytes . METHODS: Primary cultures of rat hepatocytes were treated with IL-1beta in the presence and absence of PFD . The protein and mRNA of CINC and MIP-2 were analyzed using enzyme-linked immunosorbent assays and Northern blots . RESULTS: IL-1beta increased the release of CINC and MIP-2 into culture media in a dose- and time-dependent manner . PFD inhibited both CINC and MIP-2 release in dose-dependent fashion . However, PFD had no effect on the levels of CINC mRNA induced by IL-1beta . CONCLUSION: These results suggest that PFD inhibits the production of CINC and MIP-2 by IL-1beta at a posttranscriptional step in hepatocytes. Mycopathologia, 2004 Aug, 158(2), 187 - 93 Electrophoretic variants of intracellular catalase of different Candida species; Miyasak NR et al.; Intracellular and extracellular catalases of different species of Candida were investigated using different culture media . All the Candida strains produced intracellular catalase, whose enzymatic activity was detected by non-denaturating polyacrylamide gradient (4-30%) gel electrophoresis . The cell extracts presented a major 230 kDa catalase band and in some strains variants of catalase with different molecular weights were detected . Candida catalase activity was not affected by heating at 50 degrees C and incubation with beta-mercaptoethanol, but treatment with sodium dodecyl sulphate inhibited or reduced enzymatic activity . Extracellular enzyme activity was not detected in any of the culture filtrate extracts tested. Plant Cell Rep, 2004 Nov, 23(5), 284 - 90 Epub 2004 Jul 28. In vitro germination, protocorm formation and plantlet development of mature versus immature seeds from several Ophrys species (Orchidaceae); Kitsaki CK et al.; We investigated the effect of genotype, seed maturity and culture medium on the in vitro germination and development of protocorms and plantlets from seeds of 13 different Ophrys species (O . apifera, O . attica, O . cornuta, O . delfinensis, O . ferrum-equinum, O . lutea, O . mammosa, O . speculum, O . spruneri, O . umbilicata, O . argolica, O . irricolor and O . tenthredinifera) collected in Greece, some of which are endemic to this country . Mature seeds (10 months after collection) and immature seeds (2 months after anthesis) were cultured in a coconut milk-enriched or a pineapple-enriched medium (CEM or PEM, respectively) . The highest percentage of callogenesis (96%) was observed in immature seeds of O . delphinensis in the CEM, while the highest percentage of protocorm formation (52%) was observed in mature seeds of O . spuneri in the CEM . Protocorm formation was significantly lower in immature seeds than in mature seeds in both culture media . Eventually almost all of the transferred protocorms developed to plantlets, which later formed minitubers . PEM appeared to be the most suitable for the development of minitubers from plantlets . All of the factors investigated--as well as their interactions--significantly affected callogenesis and protocorm formation . The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several under-utilized Ophrys species. ScientificWorldJournal, 2004 Oct 20, 4 Suppl 2, 83 - 90 Effects of whole-body 50-Hz magnetic field exposure on mouse Leydig cells; Forgacs Z et al.; The main goal of this study was to evaluate the possible effect of whole-body magnetic field (MF) exposure on the steroidogenic capacity of Leydig cells in vitro . In four separate experiments, male CFLP mice were exposed to sinusoidal 50-Hz, 100-microT MF . The duration of exposure was 23.5 h/day over a period of 14 days . At the end of the exposure, interstitial (Leydig) cells were isolated from the testicles of the sham-exposed and exposed animals . The cells were cultured for 48 h in the presence or absence of 1, 10, or 100 mIU/ml human chorionic gonadotropin (hCG) . The luteinizing hormone (LH) analog hCG was used to check the testosterone (T) response of the sham-exposed controls and to evaluate the possible effect of the whole-body MF exposure on the steroidogenic capacity of Leydig cells in vitro . Testosterone content of the culture media and blood sera was measured by radioimmunoassay (RIA) . In the cultures obtained from MF-exposed animals, the hCG-stimulated T response was significantly higher (p < 0.01) compared with the sham-exposed controls, while the basal T production of cells and the level of serum T remained unaltered . No MF exposure-related histopathological alterations were found in testicles, epididymes, adrenals, prostates, and pituitary glands . The MF exposure did not affect the animal growth rate and the observed hematologic and serum chemical variables . Our results indicate a presumably direct effect of whole-body MF exposure on the hCG-stimulated steroidogenic response of mouse Leydig cells. Angiogenesis, 2004, 7(2), 143 - 56 Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF; Schoenfeld J et al.; We recently published a review in this journal describing the design, hybridisation and basic data processing required to use gene arrays to investigate vascular biology (Evans et al . Angiogenesis 2003; 6: 93-104) . Here, we build on this review by describing a set of powerful and robust methods for the analysis and interpretation of gene array data derived from primary vascular cell cultures . First, we describe the evaluation of transcriptome heterogeneity between primary cultures derived from different individuals, and estimation of the false discovery rate introduced by this heterogeneity and by experimental noise . Then, we discuss the appropriate use of Bayesian t-tests, clustering and independent component analysis to mine the data . We illustrate these principles by analysis of a previously unpublished set of gene array data in which human umbilical vein endothelial cells (HUVEC) cultured in either rich or low-serum media were exposed to vascular endothelial growth factor (VEGF)-A165 or placental growth factor (PlGF)-1(131) . We have used Affymetrix U95A gene arrays to map the effects of these factors on the HUVEC transcriptome . These experiments followed a paired design and were biologically replicated three times . In addition, one experiment was repeated using serial analysis of gene expression (SAGE) . In contrast to some previous studies, we found that VEGF-A and PlGF consistently regulated only small, non-overlapping and