Culture medium

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 culture media-dependant sets of HUVEC transcripts, despite causing significant cell biological changes.

J Gene Med . 2004 Oct 28; {Epub ahead of print}
Immune responses following salivary gland administration of recombinant adeno-associated virus serotype 2 vectors; Kok MR et al.; BACKGROUND: Gene transfer to salivary glands (SGs) can be accomplished in a minimally invasive manner, resulting in stable, long-term secretion of the transgene product . Therefore, SGs provide a novel target site for several potentially useful clinical gene therapeutics applications . Previous studies have indicated that intravenous, intramuscular and intranasal administration of recombinant adeno-associated virus serotype 2 (rAAV2) vectors induce host immune responses . There are no reported studies on immune responsiveness of rAAV2 vector administration to SGs . MATERIAL AND METHODS: Vectors were administered by retrograde infusion to the SGs of Balb/c mice in various combinations . Thereafter, transgene expression was determined, and evaluations of host innate and adaptive immune responsiveness performed over a 56-day period . RESULTS: Histological examination of SGs from vector-treated mice showed no significant changes in appearance from controls, including the frequency of activated macrophage detection . There were also no differences in salivary flow rates among experimental groups . In vitro stimulation of splenocytes from mice administered rAAV2 showed elevated interferon-gamma levels in culture media . Significant titers of neutralizing antibodies to rAAV2 were detected in serum of mice following rAAV2 vector administration . While SGs could be transduced with low doses of vector it was not possible to repeat the administration and detect transduction with the same serotype at low doses . However, repeat administration was possible with an alternative serotype (rAAV4) . CONCLUSIONS: Following a single administration of rAAV2 vectors to SGs there is no significant innate immune response . However, rAAV2 vector administration to SGs results in both cellular and humoral immune responses . The latter may interfere with the efficacy of repeated rAAV2 vector administration . Copyright (c) 2004 John Wiley & Sons, Ltd.

Indian J Exp Biol, 2004 Oct, 42(10), 976 - 80
Secretion of metastasis related gangliosides by mouse B16-melanoma in circulation in vivo and in culture media in vitro; Saha S et al.; Mouse B16LuF1 melanoma cells of lower metastatic potential to lung were treated in vitro with same concentration (50 microM) of gangliosides prepared from plasma of mice bearing lung metastasis of B16LuF5, B16LuF9 or B16LuF10 melanoma cell lines of increasing metastatic potential to lung (LuF1 < LuF5 < LuF9 < LuF10) and injected to normal mice through tail vein . The number of metastatic tumor nodules formed in lung increased gradually in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment . Similarly, mouse B16LuF1 melanoma cells treated in vitro with 50 microM concentration of gangliosides prepared from spent culture media of B16LuF5, B16LuF9 or B16LuF10 cells cultured in vitro were injected to normal mice through tail vein . The number of metastatic tumor nodules formed in lung increased gradually in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment . The results indicated that metastasis-associated gangliosides present in plasma and culture media of B16-melanoma of increasing metastatic potential to lung enhanced metastatic potential of B16LuF1 cells . The increasing concentration of metastasis-associated gangliosides present in plasma and in culture media of B16-melanoma of increasing metastatic potential possibly determined increase in metastatic potential of B16LuF1-melanoma cells.

Lin Chuang Er Bi Yan Hou Ke Za Zhi, 2002 Jul, 16(7), 352 - 4
{Culture of marginal cells from guinea pig cochlear stria vascularis explants}; Zhang Y et al.; OBJECTIVE: To provide the prerequisite for further investigation of drugs ototoxicity and the probable mechanisms, explant culture technique of guinea pig cochlear strial marginal cells were established . METHOD: 26 healthy pigmented guinea pig were randomly divided into four groups according to the period of stria vascularis cultivated: 24 hours group (n = 8); 72 hours group (n = 8); more than 72 hours group (n = 8); control group (fresh stria vascularis fixed group, n = 2) . Several explants of stria vascularis and spiral ligament obtained by mechanical dissociation were cultivated and kept at 37 degrees Cwith maximal humidity in 5% CO2/95% air . The following culture media were used: E-MEM with hepes buffer (20 mmol/L), fetal calf serum (10%) . RESULT: The normal activity of stria vascularis explants cultivated for 24 hours may maintain . There were no significant change in the structure features of stria vascularis between 24-hour group and control group . There were significant difference in the structure features between 72-hour group and control group . The stria vascularis of 72-hour group cannot be observed normal stria vascularis structure, the stria vascularis structure was loose, the marginal cells reached the border of the explant and they proliferated outside the explant . The marginal cells cultivated from the stria vascularis explant may be cultivated in cell-culture dishs for 13 days . CONCLUSION: Our present results suggested that explant culture technique of cochlear strial marginal cells of guinea pigs has been successfully established . The stria vascularis cultivated for 24 hours which be maintained normal activity and structure features may be used for further investigation of drugs ototoxicity and the probable mechanisms.

Reproduction, 2004 Nov, 128(5), 623 - 8
Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares; Carnevale EM et al.; Parentage identification was used to test the developmental competence of oocytes cultured under different conditions and fertilized in vivo after oocyte transfer . Oocytes were collected transvaginally from follicles of estrous mares approximately 22 h after administration of human chorionic gonadotropin . Oocytes were cultured for approximately 16 h in one of three media, with or without addition of hormones and growth factors . Groups of three or four oocytes, cultured in different media, were transferred into the oviduct contralateral to a recipient's own ovulation . Recipients were inseminated with semen from two different stallions at 15 h before and 2.5 h after oocyte transfer . Sixteen days after transfer, embryos were recovered from uteri and submitted for parentage testing . The percentage of oocytes resulting in embryonic vesicles was nearly identical (P >0.05) for transferred oocytes (32/44, 73%) versus ovulated oocytes of recipients (9/13, 69%) . More (P <0.01) oocytes were fertilized by sperm inseminated before (35/38, 92%) versus after (3/38, 8%) oocyte transfer . Tissue culture medium (TCM)-199 was superior to equine maturation medium I (EMMI; a SOF-based medium) for culturing oocytes (P <0.05), although addition of hormones and growth factors during culture did not improve (P >0.05) development of embryos.

Oral Oncol, 2004 Nov, 40(10), 1048 - 56
NF-kappaB involvement in tumor-stroma interaction of squamous cell carcinoma; Ikebe T et al.; We investigated the interaction between tumor cells and stromal fibroblasts in tumor invasion of oral squamous cell carcinoma . Gelatin zymography showed that high levels of matrix metalloproteinase (MMP)-9 were present in the tissue of squamous cell carcinoma . When tumor cells and fibroblasts were isolated from the tissue and cultured separately, significant levels of MMP-9 were lost in the culture media of tumor cells as well as fibroblasts . When tumor cells and fibroblasts were cocultured in the presence of tumor necrosis factor alpha, high levels of MMP-9 were recovered in the culture media . The levels of MMP-9, which were secreted from tumor cells, but not fibroblasts, correlated with the number of cocultured fibroblasts . Cocultured fibroblasts, moreover, enhanced the induction of an active form of MMP-9, cell motility, and the activation of a transcription factor NF-kappaB in tumor cells . Stromal fibroblasts may induce NF-kappaB activation and promote the invasion of oral squamous cell carcinoma.

Bioorg Med Chem Lett, 2004 Dec 6, 14(23), 5731 - 3
In vitro advanced antimycobacterial screening of cobalt(II) and copper(II) complexes of fluorinated isonicotinoylhydrazones; Maccari R et al.; The in vitro antimycobacterial activity of cobalt(II) and copper(II) complexes of some fluorinated isonicotinoylhydrazones was evaluated in a TB-infected macrophage model; all metalcomplexes exhibited excellent activity against Mycobacterium tuberculosis Erdman growing within macrophages, at concentrations much lower than in culture media . Moreover complexes 1b and 2a displayed EC(99) values lower than that of the parent-drug, isoniazid . In addition, all tested metalchelates significantly inhibited the growth of single-drug-resistant M . tuberculosis strains; complexes 1a and 2a also possessed moderate activity against Mycobacterium avium complex.

Clin Exp Obstet Gynecol, 2004, 31(3), 179 - 82
A comparison of in vitro fertilization outcome by culture media used for developing cleavage-stage embryos; Summers-Chase D et al.; PURPOSE: To determine whether removing glucose and phosphate from media used for developing cleavage-stage embryos improves outcome following transfer of fresh or frozen embryos . Furthermore the study would evaluate the efficacy of adding nonessential amino acids and glutamate to media . METHODS: Embryo development was rotated on a weekly basis in human tubal fluid (HTF), versus two media relatively devoid of glucose and phosphate (e.g., P1), with one having the addition of essential amino acids and glutamate (Quinn's Advantage Medium) . RESULTS: For fresh cycles, the implantation rate was significantly higher for Quinn's . There was less fragmentation with P1 and Quinn's . For frozen cycles, the viable pregnancy, implantation rates and embryo quality were higher for Quinn's and P1 than HTF . CONCLUSION: Removal of glucose and phosphate for day-2 embryos improves in vitro fertilization outcome after embryo transfer . It is not clear if adding certain non-essential amino acids and glutamate provides further improvement.

Int J Oncol, 2004 Nov, 25(5), 1343 - 8
A novel oncoprotein RNF43 functions in an autocrine manner in colorectal cancer; Yagyu R et al.; We previously analyzed expression profiles of 20 colorectal tumors by means of genome-wide cDNA microarray . Among the genes that were commonly up-regulated in the CRCs, we further characterized biological importance of a novel human gene termed RNF43 (RING finger protein 43) in colorectal carcinogenesis . Multiple-tissue northern blot analysis revealed undetectable expression of RNF43 in normal adult tissues examined and low levels of expression in fetal kidney and lung . Its exogenous expression conferred a growth-promoting effect in COS7 and NIH3T3 cells, and suppression of its expression by specific short interfering RNAs retarded the growth of colon cancer cells . Interestingly, RNF43 protein was shown to be a secreted protein, and addition of the conditioned media of the RNF43-transfected cells into culture media of NIH3T3 cells revealed a significant enhancement of cell growth . These data suggest that RNF43 may exert its growth promoting effect in an antocrine manner, and that it may be a novel diagnostic marker for colorectal cancer.

Odontology, 2004 Sep, 92(1), 27 - 35
Heat curing of UTMA-based hybrid resin: effects on the degree of conversion and cytotoxicity; Sailynoja ES et al.; This study was designed to determine the effects of the heat curing time on a urethane tetramethacrylate (UTMA)-based hybrid resin and specifically on the degree of conversion (DC) and cytotoxicity . The materials used in this study were Estenia, a new-generation hybrid resin, and an experimental fiber reinforcement, Br-100 . The DC values of the hybrid resin samples were measured using a Fourier transform infrared (FTIR) spectrophotometer after 180 s of light curing followed by heat curing (0, 15, 30, and 60 min) . A method comparing intensities of C=C and N-H vibrations of the sample was used to calculate the final DC values . FTIR spectra were measured both inside and on the surface of the sample . The calculated DC values increased by increasing the heat curing times . After light curing only and after 15-min heat curing, the DC values inside the samples were smaller than the corresponding DC values at the surfaces of the samples . After 60 min of heat curing, the samples achieved homogeneous polymerization (DC% = 65) . The cytotoxicity of the material was studied from the glass fiber-reinforced hybrid resin samples, which were first light cured and then heat cured (15, 30, and 60 min) . Cytotoxicity was tested using both direct contact and extract methods . For the extract tests, the test specimens were incubated in a cell culture media at 37 degrees , 54 degrees , or 72 degrees C for 24 h . The heat curing times used had no effect on cytotoxicity . The incubation temperature, however, did have a significant effect . The extract obtained from 72 degrees C incubation showed a cytotoxic effect whereas the others did not . The direct contact test did not show cytotoxicity .

Rev Iberoam Micol, 2001 Sep, 18(3), 113 - 7
{In vitro susceptibility of dematiaceous fungi to ten antifungal drugs using an agar difussion test.}; Cermeno-Vivas JR et al.; We assessed the usefulness of an agar diffusion method, NeoSensitabstrade mark, to determine in vitro sensitivity of 52 isolates of dematiaceous filamentous fungi against ten antifungal agents: amphotericin B, 5-fluorocytosine, ketoconazole, fluconazole, itraconazole, terbinafine, bifonazole, miconazole, clotrimazole, and griseofulvin . For the preparation of the inoculum, a spectrophotometric method including both Shadomy and Casitone agar (CAS) culture media was used . Dematiaceous filamentous fungi were sensitive to itraconazole, terbinafine and bifonazole . Ketoconazole (90.4%), miconazole (71%), and clotrimazole (46%) showed a variable susceptibility pattern . Most species were resistant to griseofulvin and fluconazole (96%) . All isolates were resistant to 5-fluorocytosine . Sixty-three percent of strains were susceptible to amphotericin B and 28.8% resistant . Inhibition zones in the antifungal susceptibility testing did not vary according to culture medium, although fungal growth was better in CAS . Variations in antifungal sensitivity in Exophiala spinifera and Fonsecaea pedrosoi spp . would justify an in vitro susceptibility study when indicating antifungal therapy . These results show that NeoSensitabstrade mark agar diffusion method is simple, rapid, and low-cost and can be available to many clinical laboratories for the study of in vitro sensitivity of dematiceous moulds.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Oct, 24(10), 1174 - 6
{Brain-derived neurotrophin factor inhibits steroid biosynthesis by human granulosa-lutein cells.}; Chen W et al.; OBJECTIVE: To study the effect of brain-derived neurotrophin factor (BDNF) on the synthesis of estradiol and progesterone in human granulose-lutein cells (HGLCs) and the expression of steroidogenic acute regulatory factor (STAR) mRNA . METHODS: HGLCs were obtained from infertile women undergoing ovulation induction for fertilization-embryo transfer (IVF-ET) for male or tubal factors . HGLCs were cultured in serum-free media 199 for 24 h and treated by BDNF at 25, 50 and 100 ng/ml . Radio immunoassay (RIA) was used to examine the concentration of estradiol and progesterone, and reverse transcriptional PCR (RT-PCR) employed to detect the expression of STAR mRNA after treatment with BDNF at the concentrations of 25, 50 and 100 ng/ml . RESULTS: BDNF significantly inhibited the production of progesterone (P(4)) in the culture media of HGLCs in a dose-dependent manner . BDNF did not change the level of 17beta-estradiol (E(2)), but decreased the expression of STAR mRNA dose-dependently . CONCLUSIONS: BDNF can inhibit the synthesis of P(4) in HGLCs and regulate ovarian steroidogenesis . BDNF may inhibit HGLCs from producing P(4) by decreasing the transcription level of STAR gene in human ovary, and plays an important role in luteal regression.

Biochem Biophys Res Commun, 2004 Nov 19, 324(3), 1069 - 80
Similarity in cyst wall protein (CWP) trafficking between encysting Giardia duodenalis trophozoites and CWP-expressing human embryonic kidney-293 cells; Abdul-Wahid A et al.; Cyst wall proteins 1 and 2 (CWP1 and CWP2) are major constituents of the giardial cyst wall and are expressed with similar kinetics by encysting trophozoites . In the present study, we were interested to determine if the expression of giardial CWPs as heterologous proteins in a higher eukaryotic cell would result in their trafficking across the secretory pathway, as is the case in encysting trophozoites . Recombinant (r)CWP1 and rPro-CWP2 were detected in the lysate and culture media of transfected HEK-293 cells . We then conducted intracellular localization experiments using confocal microscopy and found that the proteins were trafficked in membrane enclosed vesicles across the secretory pathway and released to the culture medium by transfected HEK-293 cells . We then dissected the rCWP1 and rPro-CWP2 molecules to identify the portion(s) responsible for their secretion and found that the putative N-terminal signal peptide was sufficient for directing the secretion of rCWP1, while both the putative N-terminal signal peptide and the 13kDa C-terminal regions were necessary for the secretion of rPro-CWP2 by transfected HEK-293 cells . Taken together, these results demonstrate the degree of conservation of signal peptide recognition between lower and higher eukaryotes.

Fertil Steril, 2004 Oct, 82 Suppl 3, 1043 - 7
Synergistic effect of interleukin (IL)-1alpha and ceramide analogue on the production of IL-6, IL-8, and macrophage colony-stimulating factor by endometrial stromal cells; Kawano Y et al.; OBJECTIVE: To measure the level of interleukin 6 (IL-6), IL-8, and macrophage colony-stimulating factor (M-CSF) induced by IL-1alpha in endometrial stromal cells (ESC) following treatment with ceramide analogues . DESIGN: The effects of IL-1alpha, IL-1 receptor antagonist (IL-1RA), C2-ceramide, and C6-ceramide on the production of IL-6, IL-8, and M-CSF by ESC . SETTING: Research laboratory at Oita University Medical School . PATIENT(S): Eleven premenopausal women who had undergone hysterectomies for subserous myoma provided endometrial specimens in the secretory phase . INTERVENTION(S): The ESC were incubated for 24 hours with IL-1alpha, IL-1RA, C2-ceramide, and C6-ceramide . MAIN OUTCOME MEASURE(S): The levels of IL-6, IL-8, and M-CSF in the culture media were measured via enzyme-linked immunoabsorbent assay . RESULT(S):: Following stimulation by IL-1alpha, the production of IL-6, IL-8, and M-CSF showed a statistically significant increase, and they were suppressed by IL-1RA in a dose-dependent manner . Production of IL-6, IL-8, and M-CSF was not statistically significantly increased by IL-1alpha plus C2-ceramide as compared with IL-1alpha alone . Production of both IL-8 and M-CSF was statistically significantly increased by IL-1alpha plus C6-ceramide as compared with IL-1alpha alone; however, IL-6 production was not increased . CONCLUSION(S): The results suggest that IL-1alpha stimulates the production of IL-8 and M-CSF by a mechanism that involves the sphingomyelin-ceramide system . Ceramide may be important in increasing the production of IL-8 and M-CSF in the human endometrium.

Fertil Steril, 2004 Oct, 82 Suppl 3, 1029 - 35
Early embryo-endometrial signaling modulates the regulation of matrix metalloproteinase-3; Lahav-Baratz S et al.; OBJECTIVE: To examine matrix metalloproteinase-3 (MMP-3) expression in human stromal cell culture after P stimulation and the effect of conditioned medium from human embryo-epithelial cells coculture on its expression and activity . DESIGN: Metabolic and endocrine studies on human tissue . SETTING: In vitro fertilization (i.v.f.) unit and endocrine research unit . PATIENT(S): Infertile patients undergoing endometrial tissue sampling for dating at the luteal phase before i.v.f . INTERVENTION(S): Endometrial sampling and collection of human embryos culture media . MAIN OUTCOME MEASURE(S): Expression and activity of secreted MMP-3 by P-induced stromal cells, and in stromal cells exposed to conditioned medium from embryo-epithelial cell coculture . RESULT(S): Expression and activity of MMP-3 in human stromal cells decrease after P induction . Following incubation of these stromal-derived decidual cells with conditioned medium from embryo-epithelial cell coculture, MMP-3 expression and activity increased in a statistically significant manner . CONCLUSION(S): Progesterone inhibition of MMP-3 expression and its support of endometrial integrity were prevented by local expression of MMP-3 in response to embryonic signaling.

Fertil Steril, 2004 Oct, 82 Suppl 3, 1014 - 8
Cultured human endometrial epithelial cells produce thymus and activation-regulated chemokine with stimulation of interleukin-4 and interleukin-13; Nasu K et al.; OBJECTIVE: To evaluate the effects of T-helper (Th)1 and Th2 cytokines on the production of thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) by cultured endometrial epithelial cells (EEC) and endometrial stromal cells (ESC) . DESIGN: The effects of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-13, interferon-gamma (IFN-gamma), and tumor necrosis factor-beta (TNF-beta) on the production of TARC and MDC were investigated . SETTING: Research laboratory at a medical school . PATIENT(S): Fifteen endometrial specimens in the mid-late secretory phase were used . INTERVENTION(S): The EEC and ESC were incubated for 24 hours with recombinant human IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IFN-gamma, and TNF-beta . MAIN OUTCOME MEASURE(S): The concentrations of TARC and MDC in the culture media were measured using ELISA . RESULT(S): Small amounts of TARC were detected in the culture medium of nonstimulated EEC . The increase in levels of TARC in the culture media of EEC paralleled the addition of increasing amounts of IL-4 and IL-13 . Other cytokines, however, did not affect the production of TARC by EEC . Production of TARC by ESC was not detected under either nonstimulated or cytokine-stimulated conditions . Production of MDC was not detected in the culture media of EEC and ESC . CONCLUSION(S): These results suggest that IL-4 and IL-13 secreted from the embryo during the implantation period may selectively up-regulate the production of TARC by EEC . The controlled production of TARC in the endometrium may contribute to the modulation of the immune reaction by the regulation of Th2 lymphocyte trafficking and functions.

J Proteome Res, 2004 Sep-Oct, 3(5), 1063 - 8
Evaluation of metabolic labeling for comparative proteomics in breast cancer cells; Gehrmann ML et al.; Protein expression patterns in the cytosol of MCF-7 cells resistant to adriamycin and to adriamycin/verapamil were compared to that of the parental MCF-7 cell line and to each other using metabolic labeling and two-dimensional gel electrophoresis . Growing the parental MCF-7 cell line in 13C6-arginine- and 13C6-lysine-enriched medium resulted in C-terminal labeling of all tryptic peptides . The culture media was optimized for the incorporation of these labeled amino acids under conditions that also supported cell growth . Protein abundances were found to be distinctive in MCF-7 cells resistant to adriamycin and those selected for resistance to both adriamycin and verapamil.

Endocr Res, 2004 May, 30(2), 225 - 38
Effects of growth hormone on insulin signal transduction in rat adipose tissue maintained in vitro; Castro FC et al.; Growth hormone treatment (GH) decreases adipose tissue sensitivity to insulin . However, the exact molecular mechanism(s) involved remains unclear . In the present study, we have evaluated the chronic effects of GH on adipose tissue explants cultured in a defined media . The objective was to determine the effects of GH treatment for 24 and 48 hours on the early steps of the insulin signal transduction, including IRS-3 . The 24-hour culture media contained no hormones or 100 ng/ml GH . The 48-hour culture media contained insulin and dexamethasone supplemented with or without 100 ng/ml of GH . Results demonstrated a reduction in the cellular concentration of IRS-1 by around 30% when adipose tissue was chronically treated with growth hormone for either 24 or 48 hours . IRS-3 protein levels were also decreased by 15% after the 24-hour treatment, and by 27% after culture with GH for 48 hours in the presence of insulin and dexamethasone . PI 3-kinase concentrations were also reduced by GH in both experiments by around 25% . At the end of the 24-hour culture with GH adipose explants were stimulated with insulin in a short-term incubation, after which phosphorylation and association of the IRSs with PI 3-kinase were evaluated . After the insulin stimulus, the association of PI 3-kinase with IRS-1 and IRS-3 were decreased in explants chronically cultured with GH by 44 and 28%, respectively . After this short-term insulin stimulus, the IRS-3 phosphorylation was also lowered in GH-treated explants . The results with chronic cultures of adipose presented here are consistent with similar changes in IRS-1 and IRS-2 concentration and phosphorylation observed for liver and muscle after long-term (3-5 days) in vivo treatment with GH . The data suggest that chronic GH treatment alters the early steps of the insulin signal transduction pathway, and may explain the changes in adipose tissue sensitivity to insulin.

Inflamm Bowel Dis, 2004 Sep, 10(5), 584 - 92
Annexin 1 is secreted in situ during ulcerative colitis in humans; Vergnolle N et al.; Although annexin l exerts extracellular anti-inflammatory properties, little is known about its release in inflammatory diseases . Here, we characterized annexin 1 secretion in ulcerative colitis (UC) patients . Annexin 1 was detected by immunoblotting, in tissue homogenates and supernatants of colonic biopsies incubated in culture media, and in luminal colonic perfusates of UC patients . Annexin 1 was released by inflamed colonic biopsies from patients having severe UC but not by biopsies from healthy colon of the same patient or by biopsies from non-UC patients or from patients with slight or moderate UC . Annexin 1 was detected in luminal colonic perfusates of patients having moderate or slight UC but not in perfusates from control patients . The level of annexin 1 expression and secretion was unrelated to long-term glucocorticoid treatment, but annexin 1 secretion in perfusates was induced, in some patients, by short-term glucocorticoid exposure . These results show that annexin 1 is secreted endogenously in the colon of patients with UC . This secretion, which occurs both in vitro and in vivo, depends on the severity of inflammation . Given the anti-inflammatory effects of annexin 1, this protein may serve to down-regulate the inflammatory response in the course of inflammatory bowel disease .

Biotechniques, 2004 Sep, 37(3), 406, 408, 410 - 2
Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids; Huang KT et al.; Soluble formazan assays are widely used for cell number assessment . However, in our hands, we observed frequent occasions in which the actual cell number was at odds with the assay reading . In this study, we have determined that (i) a large proportion of the reading obtained in commonly used culture media can be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls; (ii) the albumin present in 10% serum can reduce the assay absorbance by 50% so that an actual doubling of cell number can be obscured; and (iii) this latter effect is dependent on the concentration of fatty acids . To counter these problems, we have developed a protocol that gives consistent readings that are fully representative of cell number while retaining some of the original advantages of soluble formazan assays.

J Biochem Mol Biol, 2004 Jul 31, 37(4), 454 - 9
Mitochondrial damage and metabolic compensatory mechanisms induced by hyperoxia in the U-937 cell line; Scatena R et al.; Experimental hyperoxia represents a suitable in vitro model to study some pathogenic mechanisms related to oxidative stress . Moreover, it allows the investigation of the molecular pathophysiology underlying oxygen therapy and toxicity . In this study, a modified experimental set up was adopted to accomplish a model of moderate hyperoxia (50% O(2), 96 h culture) to induce oxidative stress in the human leukemia cell line, U-937 . Spectrophotometric measurements of mitochondrial respiratory enzyme activities, NMR spectroscopy of culture media, determination of antioxidant enzyme activities, and cell proliferation and differentiation assays were performed . The data showed that moderate hyperoxia in this myeloid cell line causes: i) intriguing alterations in the mitochondrial activities at the levels of succinate dehydrogenase and succinate-cytochrome c reductase; ii) induction of metabolic compensatory adaptations, with significant shift to glycolysis; iii) induction of different antioxidant enzyme activities; iv) significant cell growth inhibition and v) no significant apoptosis . This work will permit better characterization the mitochondrial damage induced by hyperoxia . In particular, the data showed a large increase in the succinate cytochrome c reductase activity, which could be a fundamental pathogenic mechanism at the basis of oxygen toxicity.

Med J Malaysia, 2004 May, 59 Suppl B, 11 - 2
The effects of autologous human serum on the growth of tissue engineered human articular cartilage; Badrul AH et al.; Culture media supplemented with animal serum e.g . fetal bovine serum; FBS is commonly used for human culture expansion . However, for clinical application, FBS is restricted as its carry a risk of viral or prion transmission . Engineering autologous cartilage with autologous human serum supplementation is seen as a better solution to reduce the risk of transmitting infectious diseases and immune rejection during cartilage transplantation . The purpose of this study is to establish and compare the effects of 10% autologous human serum (AHS) and 10% FBS on the growth of chondrocytes and the formation of tissue engineered human articular cartilage.

Physiol Genomics, 2004 Dec 15, 20(1), 45 - 54 Epub 2004 Oct 05.
Gene expression profiling in chronic copper overload reveals upregulation of Prnp and App; Armendariz AD et al.; The level at which copper becomes toxic is not clear . Several studies have indicated that copper causes oxidative stress; however, most have tested very high levels of copper exposure . We currently have only a limited understanding of the protective systems that operate in cells chronically exposed to copper . Additionally, the limits of homeostatic regulation are not known, making it difficult to define the milder effects of copper excess . Furthermore, a robust assay to facilitate the diagnosis of copper excess and to distinguish mild, moderate, and severe copper overload is needed . To address these issues, we have investigated the effects on steady-state gene expression of chronic copper overload in a cell culture model system using cDNA microarrays . For this study we utilized cells from genetic models of copper overload: fibroblast cells from two mouse mutants, C57BL/6-Atp7a(Mobr) and C57BL/6-Atp7a(Modap) . These cell lines accumulate copper to abnormally high levels in normal culture media due to a defect in copper export from the cell . We identified 12 differentially expressed genes in common using our outlier identification methods . Surprisingly, our results show no evidence of oxidative stress in the copper-loaded cells . In addition, candidate components perhaps responsible for a copper-specific homeostatic response are identified . The genes that encode for the prion protein and the amyloid-beta precursor protein, two known copper-binding proteins, are upregulated in both cell lines.

Jpn J Clin Oncol, 2004 Sep, 34(9), 499 - 504
The synergistic cytotoxicity of cisplatin and taxol in killing oral squamous cell carcinoma; Huang GC et al.; BACKGROUND: Platinum, 5-fluorouracil (5-FU) and taxanes are commonly used in chemotherapeutic modalities of various carcinomas . However, taxanes are rarely used in patients suffering from head and neck squamous cell carcinoma (HNSCC) in Taiwan . The purpose of this study was to assess whether there is a synergistic effect produced by incorporating Taxol (paclitaxel) with cisplatin, carboplatin or 5-FU in the combined treatment of oral squamous cell carcinoma (OSCC) . METHODS: OSCC cells were surgically excised from a Taiwanese patient and cultured into a cell line, OECM-1 . The viability of OECM-1 after drug treatment was determined by an XTT labeling reagent . RESULTS: The dose-dependent cytotoxicity of each drug was determined . The order of chemosensitivity of OECM-1 toward these drugs was Taxol, cisplatin, carboplatin and 5-FU, with 50% inhibitory concentrations (IC(50)s) of 10, 68, 332 and 3000 microM, respectively . In the combined drug treatment, low concentrations of platinum (10 microM) or 5-FU (500 microM) were included in the culture media with low cytotoxic concentrations of Taxol (0.025, 0.05 and 0.1 microM) . When combined with 0.025 microM of Taxol, only cisplatin, rather than carboplatin and 5-FU, showed synergistic cytotoxicity with OECM-1 . Cisplatin also acted synergistically with 0.05 and 0.1 microM of Taxol . On the other hand, carboplatin and 5-FU acted additively with low cytotoxic concentrations of Taxol (0.025, 0.05 and 0.1 microM) . CONCLUSIONS: Our preliminary results suggest that there may be a beneficial outcome in incorporating Taxol into the chemotherapeutic modalities of HNSCC patients in Taiwan . Furthermore, at least some of the OSCC cells may be more sensitive to Taxol/cisplatin than to Taxol/carboplatin or Taxol/5-FU treatment.

Cancer Res, 2004 Oct 1, 64(19), 7078 - 85
Development of an autocrine neuregulin signaling loop with malignant transformation of human breast epithelial cells; Li Q et al.; Neuregulin (NRG) is a heparin-binding factor that activates members of the epidermal growth factor family of tyrosine kinase receptors including erbB2 that is overexpressed in more aggressive types of breast cancer . The exact role that NRG plays in breast cancer is complicated by the fact that NRG has been shown to have both proliferative and antiproliferative effects, depending on the breast cancer cell line used . Using an isogenic series of breast epithelial cell lines (MCF10A) ranging from benign to malignant, we found that the actions of NRG changed from antiproliferative to proliferative as the cells progress to cancer . This correlated with a progressive inability of NRG to down-regulate a group of proliferation genes identified previously using cDNA microarrays . As the cells progress to malignancy, they expressed higher levels of erbB2 and lower levels of erbB3 and secreted high levels of NRG into the culture media, resulting in high basal levels of erbB receptor phosphorylation . Disruption of this autocrine signaling loop by blocking ligand-induced receptor activation inhibited cancer cell proliferation . These results demonstrate that the transition of MCF10A cells from normal to premalignant to malignant correlates with the development of a constitutively active autocrine NRG signaling loop that promotes cell proliferation and suggest that disrupting this autocrine loop may provide an important therapeutic measure to control breast cancer cell growth.

Toxicology, 2004 Dec 15, 205(3), 211 - 21
Applying whole water samples to cell bioassays for detecting dioxin-like compounds at contaminated sites; Schirmer K et al.; Methodology was developed in order to rapidly and cost-efficiently screen whole water samples without extraction for the presence of dioxin-like compounds using a cell bioassay approach . Presence of dioxin-like compounds was indicated by the induction in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1, of cytochrome CYP1A, which was measured as 7-ethoxyresorufin-O-deethylase (EROD) activity . Two simple culture media, L-15/ex and Earle's-G, prepared in tissue culture water and supplemented with 5% serum, proved suitable for supporting RTL-W1 cell viability and induction of EROD activity by the model inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo{a}pyrene (BaP) . Preparation of the same simplified media using whole surface and ground water instead of tissue culture water again allowed EROD induction by spiked TCDD and BaP to be detected but higher concentrations of inducers were necessary . Despite this reduced sensitivity, RTL-W1 cells responded to 4 out of 40 ground water samples from a former oil and lignite processing site with significant EROD induction . In the future, the value of the bioassay is as an inexpensive means of quickly screening ground and surface water samples to identify high contaminant levels particularly at industrial sites, where detailed site-investigations and long-term monitoring programs are required.

Arthritis Rheum, 2004 Sep, 50(9), 2839 - 48
Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS-4 (aggrecanase 1) or ADAMTS-5 (aggrecanase 2); Chockalingam PS et al.; OBJECTIVE: To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin-1 (IL-1) or retinoic acid or in vivo in synovial joints during aging and joint pathology . METHODS: Bovine articular cartilage discs (live or freeze-killed) were cultured in the presence of IL-1 or were incubated in digestion buffer containing recombinant human ADAMTS-4 (rHuADAMTS-4; aggrecanase 1) or rHuADAMTS-5 (aggrecanase 2) . Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanase-generated G1 domain (using neoepitope monoclonal antibody AGG-C1/anti-NITEGE(373)) and link proteins (using monoclonal antibody 8-A-4), as well as by quantitative enzyme-linked immunosorbent assays to detect aggrecanase-generated G1 domain (G1-NITEGE(373)) and HA . RESULTS: IL-1 treatment of live cartilage explants induced a time-dependent release of sGAG, aggrecanase-generated G1 domain (G1-NITEGE(373)), and HA into the culture media . Exposure of live or freeze-killed articular cartilage discs to rHuADAMTS-4 or rHuADAMTS-5 resulted in a dose- and time-dependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1-NITEGE(373) and link proteins) . CONCLUSION: Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

Rev Iberoam Micol, 2003 Dec, 20(4), 172 - 5
{A survey of temperature and pH effect on colonial growth of Botryodiplodia theobromae RC1}; Eng F et al.; Study of fungal colonial growth is a basic method to examine their behaviour in different cultivation conditions . The influence of temperature and initial pH on growth radial velocity and growth density of Botryodiplodia theobromae RC1, was studied in order to show the growth characteristics of this fungus . Both temperature and culture medium influenced growth density, but radial velocity of growth was only affected by temperatures above 40 degrees C . In addition, initial pH of culture media did not affect either parameter.

Wound Repair Regen, 2004 Sep-Oct, 12(5), 557 - 64
Expression of transforming growth factor-beta and extracellular matrix by human peritoneal mesothelial cells and by fibroblasts from normal peritoneum and adhesions: effect of Tisseel; Saed GM et al.; We have previously shown that fibroblasts obtained from adhesions produce greater amounts of transforming growth factor-beta 1 (TGF-beta1) and extracellular matrix (ECM) molecules than normal fibroblasts isolated from normal peritoneum . The purpose of the current studies was to examine the effect of Tisseel (Baxter Healthcare Corporation, Glendale, CA), a fibrin sealant containing fibrinogen, aprotinin (a protease inhibitor), thrombin, and CaC1(2), on TGF-beta1 and ECM production by human peritoneal mesothelial cells, normal peritoneal fibroblasts, and adhesion fibroblasts . Multiplex reverse transcription-polymerase chain reaction using beta-actin as a housekeeping gene was used to determine mRNA levels of TGF-beta1 and ECM in these cells at 6, 12, 24, and 48 hours under normoxic conditions in the following treatment groups : fibrin sealant (Tisseel) alone; fibrin sealant with the two components diluted 1 : 2; fibrin sealant with the sealer protein component reconstituted without aprotinin (a protease inhibitor); fibrin sealant with the sealer protein component reconstituted without aprotinin (and both components diluted 1 : 2); fibrin sealant components diluted to physiologic concentrations; and control (culture media) . The test compositions had little effect on TGF-beta1 mRNA expression in mesothelial cells and normal peritoneal fibroblasts, but resulted in a marked reduction of TGF-beta1 from adhesion fibroblasts . Expression of type I collagen by human peritoneal mesothelial cells was not detected; the compositions reduced type I collagen mRNA expression by both types of fibroblasts . Type III collagen was detected at six hours, and increased approximately 50 percent by culturing for 48 hours . Tisseel at full strength and with both components diluted 1 : 2 initially increased type III collagen mRNA levels; in contrast, type III collagen mRNA levels were reduced in mesothelial cells by the fibrin sealant without aprotinin at both concentrations and at physiologic concentrations . In both types of fibroblasts, the Tisseel compositions reduced type III collagen mRNA expression . Fibronectin mRNA were transiently reduced at six hours by approximately 50 percent in the presence of the Tisseel components, but then returned to control levels . Fibronectin mRNA levels were not altered in normal peritoneal fibroblasts, but were reduced by all but the physiologic concentration in adhesion fibroblasts . Tisseel may modulate human peritoneal mesothelial cell, normal peritoneal fibroblast, and adhesion fibroblast function . These results suggest that fibrin sealant prepared from the Tisseel kit without aprotinin has the ability to reduce ECM and TGF-beta1 mRNA levels, especially from adhesion fibroblasts, which may indicate a role in reduction of postoperative adhesion development.

Theriogenology, 2004 Nov, 62(8), 1403 - 16
Effect of protein supplementation in potassium simplex optimization medium on preimplantation development of bovine non-transgenic and transgenic cloned embryos; Bhuiyan MM et al.; The present study evaluated the effect of protein supplementation in potassium simplex optimization medium (KSOM) on bovine preimplantation embryo development . The in vitro fertilized (IVF) (Experiment 1), non-transgenic (Experiment 2) and transgenic cloned embryos (Experiment 3) were cultured for 192 h in KSOM supplemented with 0.8% BSA (KSOM-BSA), 10% FBS (KSOM-FBS) or 0.01% PVA (KSOM-PVA) . Transfected cumulus cells with an expression plasmid for human alpha1-antitrypsin gene and a green fluorescent protein (GFP) marker were used to produce transgenic cloned embryos . Modified synthetic oviductal fluid (mSOF) supplemented with 0.8% BSA (mSOF-BSA) was used as a control medium . In Experiment 1, cleavage rate was significantly (P < 0.05) lower (69.1%) in IVF embryos cultured in KSOM-FBS than in KSOM-BSA (80.3%) . The rate of hatching/hatched blastocyst formation was significantly (P < 0.05) lower in embryos cultured in KSOM-PVA than in KSOM-FBS (2.2% versus 10.8%) . Blastocysts cultured in KSOM-FBS contained significantly (P < 0.06) higher numbers of inner cell mass cells (50.4 +/- 20.2) than those cultured in mSOF-BSA (36.9 +/- 19.2) . In Experiment 2, the rate of blastocyst formation was significantly (P < 0.05) lower (20.5%) in embryos cultured in KSOM-PVA than in other culture media (33.3-38.5%) . The rate of hatching/hatched blastocysts was significantly (P < 0.05) lower in KSOM-PVA (13.9%) and KSOM-FBS (17.1%) than in KSOM-BSA (30.8%) and mSOF-BSA (33.9%) . The numbers of total and trophectoderm cells (104.6 +/- 32.2 and 71.7 +/- 25.5, respectively) were significantly (P < 0.05) lower in blastocysts cultured in KSOM-PVA than in KSOM-BSA (125.7 +/- 39.7 and 91.7 +/- 36.2, respectively) . In Experiment 3, no significant differences in embryo development, GFP expression and blastocyst cell numbers were observed among the culture groups . In conclusion, the present study demonstrated that KSOM and mSOF supplemented with BSA were equally effective in supporting development of bovine non-transgenic and transgenic cloned embryos . Moreover, different developmental competence in response to protein supplementation of KSOM was observed between bovine non-transgenic and transgenic cloned embryos.

Mycol Res, 2004 Aug, 108(Pt 8), 926 - 32
Effects of culture media and environmental factors on mycelial growth and pycnidial production of Potebniamyces pyri; Xiao CL et al.; Potebniamyces pyri (anamorph Phacidiopycnis piri) is the causal agent of Phacidiopycnis rot of apples and pears . The disease has recently been recognized in pears in the USA . Little information on the basic biology of the fungus is available . In this paper, we report the effects of culture media, temperature, water potential and pH on mycelial growth, and the effects of media and light on pycnidia production . Prune juice agar was the best for rapid mycelial growth . Pear juice agar, apple juice agar, potato dextrose agar, and oatmeal agar (OMA) also favoured mycelial growth . Czapek-Dox agar was not suitable for mycelial growth . The fungus was able to grow at temperatures from -3 to 25 degrees C . Optimal mycelial growth occurred between 15 and 20 degrees . The average radial growth rate on OMA was 3.9 mm d(-1) at 15 degrees and 4.4 mm d(-1) at 20 degrees . Mycelial growth was not observed after 10 d at 30 degrees, but growth resumed at 20 degrees . The fungus failed to resume growth at 20 degrees after being incubated for 10 d at 35 degrees . The fungus was able to grow at water potentials as low as -4 MPa but no growth took place at -7.3 MPa . Active mycelial growth was observed on OMA at pH between 3.2 and 6.1 . Optimal growth was observed at pH around 4 and no growth was observed at pH 7.1 . No pycnidia or very few formed on the nine media at 20 degrees in the dark . Fluorescent light significantly stimulated formation of pycnidia . OMA was the best medium for production of pycnidia and macroconidia . Pycnidia that formed on 8-wk-old OMA cultures incubated at 20 degrees under 12 h dark/12 h light produced abundant macroconidia and the technique is recommended for inoculum production of conidia for research.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Sep, 24(9), 991 - 4
{Role of intron and 5' untranslated region in human thrombopoietin gene expression}; Ning YS et al.; OBJECTIVE: To study the role of 5' untranslated region (UTR) and intron in the expression of human thrombopoietin (TPO) gene . METHODS: A number of expression vectors containing TPO mini-gene fused to the regulatory elements of cytomegalovirus (CMV) were constructed and transfected via lipofectin into cultured cos-1 cells for transient expression of TPO gene . The cell culture media were analyzed with highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) 48 h after the transfection . RESULS: The expression levels of the TPO gene elements followed the order of TPO intron v> TPOcDNA> TPO intron I> TPO intron I> TPO gDNA in cos-1 cells . CONCLUSION: The last intron of TPO gene obviously enhances the expression level of TPO gene, which can be inhibited by 5'UTR of TPO gene.

Mycol Res, 2004 Jul, 108(Pt 7), 823 - 7
Production of gametangia by Phytophthora ramorum in vitro; Brasier C et al.; Until now gametangia have not been obtained between paired European A1 and American A2 isolates of Phytopthora ramorum in vitro . Their production in artificial culture relies on interspecific pairings . Using P . drechsleri and P . cambivora testers, 51 of 110 P . ramorum isolates from across Europe were all shown to be A1s; while 32 of 38 American isolates from across California and southwest Oregon were shown to be A2s . However, these interspecific pairings are complex, unusually slow and unpredictable . A range of culture media and conditions are described that were tested, unsuccessfully, with a view to enhancing the efficiency of the interspecific pairings . In further tests, gametangia were obtained between A1 and A2 isolates of P . ramorum when juvenile, pre-chlamydospore producing mycelia were mixed together on carrot agar . The gametangia formed in 3-10 d, sparsely to frequently, initially only within the boundaries of the mixed inocula but subsequently in the extended mycelial growth . Chlamydospores were also produced . This inoculum-mixing method, though again sometimes unpredictable, should enhance efficiency of testing for compatibility types and facilitate further studies on whether the sexual outcrossing system of P . ramorum is functional . Differences between sexual reproduction of P . ramorum and that of other heterothallic Phytophthora species are discussed.

Zhonghua Gan Zang Bing Za Zhi, 2004 Sep, 12(9), 552 - 3
{Establishment and the significance of a cell model of secreted alkaline phosphatase co-controlled by HCV 5'NCR and NS3 serine protease}; Liu SP et al.; OBJECTIVE: To establish a cell model of secreted alkaline phosphatase (SEAP) co-controlled by HCV 5'NCR and NS3 serine protease in an effort to develop new antiviral agents . METHODS: The fragments of HCV 5'NCR and NS3/4A-SEAP were amplified by PCR . They were fused into pBluescript SK+ to generate 5'NCR-NS3/4A-SEAP chimeric plasmid . The resulting chimeric gene was subcloned into HindIII/Bsu36 I site of pSEAP2-Control (a SEAP eukaryotic expression plasmid), to generate pNCR-NS3/4A-SEAP, in which the SEAP was fused in-frame to the downstream of NS4A/4B cleavage site . The SEAP activity in the culture media of transiently transfected cells was monitored quantitatively . The regulatory effect of HCV 5'NCR and NS3 serine protease on SEAP expression was measured by treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) against HCV 5'NCR and TPCK, a irreversible serine protease inhibitor . RESULTS: The SEAP activity in the culture media reached 80801+/-4794 RLU, and was significantly inhibited by 5 micromol/L, 10 micromol/L of ASODN (t=4.315, p<0.01; t=6.985, p<0.001) and 100 micromol/L of TPCK (t=6.949, P<0.001) . CONCLUSION: A cell model of SEAP co-controlled by HCV 5'NCR and NS3 serine protease has been successfully established . This might promote the screening of anti-viral drugs

J Spinal Disord Tech, 2004 Oct, 17(5), 423 - 8
Effect of bone morphogenetic protein-2 (BMP-2) on matrix production, other BMPs, and BMP receptors in rat intervertebral disc cells; Li J et al.; OBJECTIVE: An in vitro experiment study using rat disc cells was carried out to determine the effect of bone morphogenetic protein-2 (BMP-2) on extracellular matrix production, other BMPs, and BMP receptors (BMPRs) . METHODS: Cells from the anulus fibrosus and transition zone were harvested and cultured . When the cells reached 80% confluence, BMP-2 was added to reach a final concentration of 200 ng/mL . Three days later, the culture media were collected for the assay of sulfated glycosaminoglycans (sGAG) and collagen types I and II . The cells were harvested for RNA extraction to determine the genes expressed . All experiments were performed at least three times to ensure repeatability . RESULTS: BMP-2 significantly increased aggrecan and collagen type II mRNA expression 8.30 and 4.61 times, respectively, and decreased versican mRNA expression 0.54 times as compared with control . Collagen type I production and mRNA level were not changed . BMP-2 significantly increased transforming growth factor-beta1 (TGFbeta1) and BMP-7 mRNA expression 2.32 and 2.45 times, respectively, compared with control . There was no significant change in BMP-6 mRNA expression . BMPR type IB and II mRNA expressions were increased and BMPR type 1A mRNA expression was decreased, but none of these differences was significant . CONCLUSIONS: The results of this study show that in rat intervertebral disc cells, BMP-2 increases aggrecan and collagen type II mRNA expression and decreases versican gene expression . BMP-2 also up-regulates mRNA expression for BMP-7 and TGFbeta but has no significant effect on the BMPRs.

Biol Reprod, 2005 Jan, 72(1), 179 - 87 Epub 2004 Sep 22.
Similar Effects of Osmolarity, Glucose, and Phosphate on Cleavage past the 2-Cell Stage in Mouse Embryos from Outbred and F1 Hybrid Females; Hadi T et al.; One-cell-stage embryos derived from most random-bred and inbred female mice exhibit an in vitro developmental block at the two-cell stage in classical embryo culture media . However, embryos derived from many F(1) hybrids develop easily past the two-cell stage under the same conditions . This has given rise to the commonly accepted idea that there exist blocking and nonblocking types of female mice, with only the former being prone to a two-cell block . Recently, culture media have been improved to the point that even embryos prone to the two-cell block will develop past the block in vitro, making it possible to study its etiology . Here, we show that either increased osmolarity or increased glucose/phosphate levels induced the expected two-cell block in random-bred CF1 embryos and the two-cell block at increased osmolarities could be rescued by the organic osmolyte glycine . Surprisingly, one-cell embryos from B6D2F(1) (BDF(1)) F(1) hybrid females, considered to be nonblocking, also became blocked at the two-cell stage when osmolarity or glucose/phosphate levels were increased . They were also similarly rescued by glycine from the osmolarity-induced block . The most evident difference was that the purportedly nonblocking embryos became blocked at a higher threshold of osmolarity or glucose/phosphate level than those considered prone to this developmental block . Thus, both blocking and nonblocking embryos actually exhibit a similar two-cell block to development.

J Med Food, 2004 Fall, 7(3), 320 - 6
Effect of retinoic acid on leptin, glycerol, and glucose levels in mature rat adipocytes in vitro; Hong SE et al.; To elucidate the effects of retinoic acids (RAs) on adipogenesis and insulin sensitivity, we treated mature adipocytes with two different kinds of RA, 9-cis-RA and all-trans-RA . Both 9-cis- and all-trans-RA inhibited the secretion of leptin . However, the inhibition was significantly decreased at a higher dose of each RA . The inhibitory effect of 9-cis-RA was synergistically enhanced by the addition of rosiglitazone, a synthetic ligand for peroxisome proliferator-activated receptor (PPAR) gamma . 9-cis-RA also leads to adipogenesis in a dose-dependent manner . On the contrary, all-trans-RA does not increase adipogenesis in a dose-dependent manner . To clarify the antidiabetic effects of RA, glucose uptake was assessed by estimating glucose concentrations in the medium . 9-cis-RA reduced glucose levels in the culture media, but all-trans-RA did not . In conclusion, all-trans-RA does not alter adipogenesis and glucose uptake but does inhibit leptin secretion . 9-cis-RA, however, seems to increase both adipogenesis and glucose uptake through activation of the retinoid X receptor/PPARgamma heterodimer.

Chemistry, 2004 Oct 25, 10(21), 5467 - 72
Aldolase antibody activation of prodrugs of potent aldehyde-containing cytotoxics for selective chemotherapy; Sinha SC et al.; Prodrugs of potent aldehyde analogues of the anticancer drug doxorubicin (Dox) were synthesized . These prodrugs were efficiently activated by antibody 93F3 and no drug formation was observed in the absence of 93F3 in either phosphate buffered saline or cell culture media . In the presence of antibody 93F3, these prodrugs were activated and decreased the proliferation of human cancer cells in in vitro proliferation assays.

Oecologia, 2004 Nov, 141(3), 395 - 401 Epub 2004 Aug 03.
Filamentous cyanobacteria, temperature and Daphnia growth: the role of fluid mechanics; Abrusan G; Viscosity increases significantly with a fall in water temperature, thus temperature change affects not only the metabolic rates of aquatic suspension feeders, but also the physical properties of the surrounding fluid . This mechanistic effect of water temperature change on growth was separated from the effect of metabolism by using culture media with modified viscosity, while the temperature was kept constant . The effect of water viscosity on growth rate and feeding of four Daphnia species (D . magna, D . pulicaria, D . hyalina, D . galeata) was investigated . Increased viscosity decreased the growth rate significantly for three species, with the exception of D . galeata . Changing viscosity also affects growth qualitatively: the filamentous blue-green Cylindrospermopsis raciborskii reduces the growth rate of D . pulicaria at low viscosity, but its negative effect disappears when viscosity is higher . The findings are consistent with the hypothesis that it is the Reynolds number of the filtering appendages that determines the qualitative features of Daphnia filtration . The edibility of C . raciborskii at high water viscosity is most probably caused by lack of interference with filtering combs, and explains the coexistence of D . pulicaria with filamentous blue-green species in the field, and also the observed temperature dependence of growth inhibition of filaments.

Fertil Steril, 2004 Sep, 82(3), 756 - 9
Hypoxia simultaneously inhibits endostatin production and stimulates vascular endothelial growth factor production by cultured human endometrial stromal cells; Nasu K et al.; Hypoxia downregulated the concentration of endostatin in the culture media of human endometrial stromal cells but did not affect the messenger (m)RNA expression of collagen XVIII . Both mRNA and protein expression of vascular endothelial growth factor were upregulated in a hypoxic condition.

Fertil Steril, 2004 Sep, 82(3), 593 - 600
Differential growth of human embryos in vitro: role of reactive oxygen species; Bedaiwy MA et al.; OBJECTIVE: To examine the relationship of early human embryonic development with the level of reactive oxygen species (ROS) in the culture media on the first day (day 1 ROS) after insemination . DESIGN: A prospective study . SETTING: Patients undergoing assisted reproduction in a teaching hospital . PATIENT(S): Patients undergoing conventional IVF (n = 104; 115 cycles) and intracytoplasmic sperm injection (ICSI) (n = 91; 96 cycles) were included . Both fertilization and early cultures were performed in human tubal fluid with 5% serum substitute supplement . INTERVENTION(S): Day 1 ROS levels in the central well (sample) and the outer well (control) of each embryo culture dish were measured after overnight incubation by chemiluminescence assay using luminol as the probe . MAIN OUTCOME MEASURE(S): Fertilization rate and embryo quality at day 3 and 5 were recorded for each cycle . Age, parity, and demographic features were also compared . RESULT(S): High day 1 ROS levels in culture media were associated with low blastocyst rate, low fertilization rate, low cleavage rate, and high embryonic fragmentation with ICSI but not with conventional IVF . High day 1 ROS levels in culture media were associated with lower pregnancy rates in both IVF and ICSI cycles . CONCLUSION(S): Reactive oxygen species generated in culture media by day 1 may be an important biochemical marker for early embryonic growth . Increased embryonic fragmentation and slow cleavage rate may be partially attributed to early exposure of embryos to high ROS levels in ICSI cycles . Differential growth of ICSI embryos incubated under identical conditions may be in part due to differences in ROS levels of the culture medium surrounding these embryos.

Pediatr Surg Int, 2005 Jan, 21(1), 29 - 33
Glutamine regulates amino acid transport and glutathione levels in a human neuroblastoma cell line; Soh H et al.; Both amino acid transport and glutathione play a key role in regulating cancer cell growth . Glutamine can serve as an important ATP source for cancer cells, and it can supply glutamate, a precursor for the synthesis of glutathione, by the hydrolysis of glutamine . We examined the effects of glutamine concentrations {2 mM (control), 400 microM, 200 microM, and 0 microM} on cell growth, amino acid transport, and glutathione levels in a human neuroblastoma cell line, SK-N-SH, by using cell culture technique . Cell growth rates were dependent on glutamine concentrations in culture media . Glutamate transport significantly increased in glutamine-deprived groups, and this increase was remarkable in lower glutamine groups (200 microM and 0 microM glutamine) . Glutamine deprivation resulted in a significant decrease in glutathione levels by 20% compared with control, but glutathione in 0 microM glutamine was maintained with the same levels found in 400 microM and 200 microM glutamine . DNA and protein synthesis correlated directly with glutamine concentrations in culture media . Our results suggest that glutamine mediates neuroblastoma cell proliferation by regulating amino acid transport and glutathione synthesis, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply.

Biol Reprod, 2005 Jan, 72(1), 107 - 18 Epub 2004 Sep 15.
Follicle-stimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro; Roberts R et al.; Follicle-Stimulating Hormone (FSH) at a wide range of doses is routinely added to culture media during in vitro maturation (IVM) of oocytes, but the effects on oocyte health are unclear . The suggestion that superovulation may cause aneuploidy and fetal abnormalities prompted us to study the potential role of FSH in the genesis of chromosomal abnormalities during meiosis I . Mouse cumulus-oocyte complexes (COCs) isolated from the antral follicles of unprimed, sexually immature B6CBF(1) mice were cultured in increasing concentrations of FSH . Following culture, matured oocytes were isolated, spread, stained with DAPI, and the numbers of chromosomes counted . Significantly increased aneuploidy, arising during the first meiotic division, was observed in metaphase II oocytes matured in higher concentrations of FSH (>/=20 ng/ml) . The effect of FSH on spindle morphology and chromosome alignment during metaphase I was then explored using immunocytochemistry and three-dimensional reconstruction of confocal sections . High FSH had no effect on gross spindle morphology but did alter chromosome congression during prometaphase and metaphase, with the spread of chromosomes across the spindle at this time being significantly greater in oocytes cultured in 2000 ng/ml compared with 2 ng/ml FSH . Analysis of three-dimensional reconstructions of spindles in oocytes matured in 2000 ng/ml FSH shows that chromosomes are more scattered and farther apart than they are following maturation in 2 ng/ml FSH . These results demonstrate that exposure to high levels of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities and highlights the importance of the judicious use of FSH during IVM.

J Int Acad Periodontol, 2004 Jul, 6(3), 81 - 8
Modulating effect of serum on the stimulation of plasminogen activator inhibitor 2 production in human gingival fibroblasts by lipopolysaccharide and interleukin-1beta; Xiao Y et al.; OBJECTIVE: Plasminogen activator inhibitor-2 (PAI-2) is an important counter proteolysis factor which helps protect tissues from inflammatory stress . The expression of PAI-2 can be modulated by various inflammatory stimulants and mediators . The aim of the present study was to investigate how serum factors, might modulate the effects of lipopolysaccharide (LPS) and interleukin-1beta on PAI-2 production by human gingival fibroblasts . METHODS: Human gingival fibroblasts were exposed to LPS derived from Actinobacillus actinomycetemcomitans or Escherichia coli and a commercial source of interleukin-1beta (IL-1beta) . The expression of PAI-2 and its mRNA was monitored by Western blotting, RT-PCR, and Northern blotting . RESULTS: The results showed that the distribution of PAI-2 synthesised by human gingival fibroblasts (HGF) was mostly as an intracellular protein (47kDa) . The presence of serum in the culture media was absolutely necessary for both the secretion of PAI-2 and for the effect of the inflammatory mediators (LPS and IL-1beta) . A pattern of PAI-2 response in HGF after LPS stimulation was detected with a quick up-regulation of the PAI-2 mRNA, which was down regulated when extracellular PAI-2 (60kDa mass) levels reached plateau levels . The synthesis of PAI-2 by HGF, in terms of mRNA expression and protein synthesis, was more sensitive to stimulation with lower concentrations of LPS (10 ng/ml) than with higher LPS concentrations . CONCLUSIONS: PAI-2 is a serum dependent molecule with major cytosolic localisation in HGF . Its cellular accumulation and secretion can be modulated by bacterial LPS and IL-1beta through serum factors.

Hepatology, 2004 Aug, 40(2), 394 - 402
Interferon alpha-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor beta(1); de Lujan Alvarez M et al.; In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci . The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci . Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6) . Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats . Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4 . Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated . To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b . IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes . Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b . IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity . They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling . When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked . In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci .

Reprod Domest Anim, 2004 Oct, 39(5), 356 - 60
Effects of retinol on the in vitro development of Bos indicus embryos to blastocysts in two different culture systems; Lima PF et al.; The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF . A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h . In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium . Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups . Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2) . Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2) . In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed . Afterwards embryos were kept in culture or co-culture for further 9 days . In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF) . In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM) . The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts . The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.

J Pak Med Assoc, 2004 Jun, 54(6), 301 - 5
Effects of modified sample collection technique on fungal culture yield: nail clipping/scraping versus microdrill; Qureshi HS et al.; OBJECTIVE: Onychomycosis requires accurate diagnosis but fungal culture yield is frequently low by routine sampling techniques . The aim of this study was to investigate the utility of nail plate/subungual microdrilling as an alternative to conventional nail clipping/subungual scraping . METHODS: Patients with clinical evidence of onychomycosis (n=46) were prospectively evaluated for fungal potassium hydroride (KOH) microscopy and culture comparing two sampling techniques: nail clipping versus microdrilling . RESULTS: Fungal cultures were positive in 48% with 2 additional cases detected by combining both methods . KOH microscopy was positive in 17% cases . Specimen obtained via the microdrill technique gave consistent heavier fungal growth on culture media . Candida species were the most common isolates (82.7% of cases) and were negative on KOH microscopy in 95 % of culture proven cases . The microdrill technique yielded consistent heavier growth on culture media CONCLUSION: Microdrill technique improves laboratory diagnosis and ultimately treatment of onychomycosis, particularly in patients with repeated KOH microscopy and culture failure despite strong clinical suspicion.

Eur J Endocrinol, 2004 Sep, 151(3), 333 - 41
Mutations in the NSD1 gene in patients with Sotos syndrome associate with endocrine and paracrine alterations in the IGF system; De Boer L et al.; OBJECTIVE: To investigate the effect of nuclear receptor Su-var, 3-9, enhancer of zeste, trithorax (SET) domain-containing protein 1 (NSD1) gene alteration in patients with Sotos syndrome on plasma IGFs and IGF-binding proteins (IGFBPs), as well as on the IGF/IGFBP system activity at the tissue level . DESIGN: Twenty-nine patients suspected of Sotos syndrome were divided into two groups: patients with heterozygous deletions or mutations in the NSD1 gene (NSD1(+/-)) (n=11) and subjects without (NSD1(+/+)) (n=18) . Plasma samples (n=29) and skin fibroblasts (n=23) were obtained . The results of both groups were compared and related to reference values . METHODS: IGF-I, IGF-II, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-6 levels were determined by RIAs . The mitogenic response of fibroblasts to IGFs was investigated by {methyl-(3)H}thymidine incorporation . IGFBP-3 levels in the culture media were measured by RIA . IGFBP-3 mRNA expression was determined by real time RT-PCR . RESULTS: NSD1(+/-) patients showed significantly altered levels of IGF-I (mean-1.2 SDS), IGF-II (-1.2), IGFBP-3 (-1.7), IGFBP-4 (-0.4), IGFBP-2 (+0.8) and IGFBP-6 (+1.5) . The NSD1(+/+) patients did not differ from the reference, with the exception of the mean IGFBP-3 level (-1.3) . Basal proliferation and mitogenic response to IGFs was diminished in NSD1(+/-) fibroblasts compared with NSD1(+/+) (basal, P=0.02; IGF-I, P<0.001; IGF-II, P=0.02) . Compared with control fibroblasts, only the mitogenic response was diminished (basal, P=0.07; IGF-I, P=0.04; IGF-II, P=0.04) . A trend of higher IGFBP-3 secretion after IGF-I stimulation (P=0.09) and 3.5-5 times higher mRNA expression of IGFBP-3 in basal conditions was found in NSD1(+/-) fibroblasts in comparison to controls . CONCLUSIONS: NSD1(+/-) patients show endocrine and paracrine changes in the IGF system . These changes may contribute to the abnormal growth pattern.

Arterioscler Thromb Vasc Biol, 2004 Nov, 24(11), 2155 - 61 Epub 2004 Sep 09.
Cyclosporin A traps ABCA1 at the plasma membrane and inhibits ABCA1-mediated lipid efflux to apolipoprotein A-I; Le Goff W et al.; OBJECTIVE: ABCA1 mediates cellular cholesterol and phospholipid efflux to apolipoprotein A-I and other apolipoprotein acceptors . In this study, we analyzed the effect of the immunosuppressant cyclosporin A on the ABCA1-mediated lipid effluxes reactions . METHODS AND RESULTS: Cyclosporin A acted as a potent inhibitor of ABCA1 activity in several cell lines . Using the RAW264.7 mouse macrophage cell line, in which ABCA1 and its associated cholesterol efflux activity are inducible by cAMP analogues, cyclosporin A inhibition of cholesterol efflux to apolipoprotein A-I was rapidly reversible after its removal from the culture media, implying that ABCA1 levels were not drastically reduced by cyclosporin A . In fact, cyclosporin A treatment decreased ABCA1 turnover and yielded a 2-fold increase in cell-surface ABCA1 . Despite the increase in cell-surface ABCA1, cyclosporin A decreased apolipoprotein A-I uptake, resecretion, and degradation in RAW cells . Finally, consistent with the inhibition of ABCA1 in vitro, cyclosporin A treatment induced a 33% reduction of high-density lipoprotein (HDL) levels in mice . CONCLUSIONS: ABCA1 inhibition by cyclosporin A supports a role for ABCA1 endocytic trafficking in ABCA1-mediated lipid efflux and could explain in part the low HDL levels observed in some patients with transplants.

Eur J Obstet Gynecol Reprod Biol, 2004 Oct 15, 116(2), 196 - 200
A randomized control comparison study of culture media (HTF versus P1) for human in vitro fertilization; Artini PG et al.; OBJECTIVE: It is now widely accepted that increasing the number of replacement embryos (>3 embryos per embryo transfer {ET}) is associated with an increased risk of multiple pregnancies . While embryo reduction is often proposed when there is a high risk of multiple pregnancies, it is a difficult decision for the couple . For this reason, different studies have focused on single embryo transfer, more precisely blastocyst transfer . The aim of the study is to confirm that phosphate-free culture media can be used to generate greater quality embryos . METHODS AND RESULTS: We carried out a study to compare the efficacy of human tubal fluid (HTF) versus preimplantation stage one (P1) as culture media for assisted reproductive therapy (ART) . In 109 nonselected patients, we obtained an embryo fertilization rate with HTF and P1 culture media of 58.6 and 62.5% (P = 0.003), respectively . After 48 and 72 h, the morphology was similar for both P1 and HTF embryos in most patients . However, in the same patients, when HTF embryo quality was low (15.4%), P1 embryo quality was significantly higher 68.7% (P = 0.002) . Some embryos were transferred at 48 h and some at 72 h after retrieval, in a randomized manner . We transferred a maximum of up to three embryos per ET . The implantation rate was significantly different; at 48 h, it was 6.8 and 12.2% for HTF and P1, respectively (P = 0.02) . The pregnancy rate was 17.1% for HTF embryos and 23.7% for P1 embryos (P = 0.02) . CONCLUSIONS: Therefore, we observed a significant difference between P1 and HTF in the fertilization rate, in embryo quality, in implantation rate and in pregnancy rate . But the most important difference between this study and others is that every patient was the control of herself, so we eliminated every variable.

Protein Expr Purif, 2004 Oct, 37(2), 443 - 9
Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain; Razzera G et al.; NOR-1 is an orphan member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, fatty acids, retinoic acids, and other lipophilic molecules . The NOR-1 subfamily (NR4), composed also of Nurr1 and Nurr77, has been implicated in cell proliferation, differentiation, apoptosis, chondrosarcomas, inflammation, and atherogenesis . The NOR-1 receptor is an orphan ligand receptor which acts over gene transactivation . No ligands, if such in fact exist, are known for this receptor . Recently, the three-dimensional structure of the homolog receptor Nurr1 has been solved using protein crystallography techniques . Surprisingly, the structure does not present either a typical cavity for ligand binding or a classical co-factor binding site in the ligand binding domain (LBD) . To allow for structural studies of other members of NR4 subfamily, we have subcloned, overexpressed in Escherichia coli cells, purified, and characterized the rat orphan nuclear receptor NOR-1 LBD domain . We obtained NOR-1 LBD at a high degree of purity and with an overall yield of 3 mg/L of culture media . CD spectroscopic analysis shows a high alpha-helical secondary structure content (52%), similar to that of Nurr 1 LBD three-dimensional structure . Thermal denaturation monitored by UV absorption and CD spectroscopy suggests proper folding of recombinant NOR-1 LBD.

Pigment Cell Res, 2004 Oct, 17(5), 506 - 14
Pheomelanin production in the epidermis from newborn agouti mice is induced by the expression of the agouti gene in the dermis; Hirobe T et al.; The present study was designed to clarify the role of the agouti gene in the regulation of the proliferation and differentiation of mouse epidermal melanocytes using serum-free primary culture of epidermal melanocytes from 0.5-d-old black (a/a; C57BL/10JHir) mice and congenic, agouti (A/A; C57BL/10JHir-A/A) mice . There was no significant difference in the proliferation or differentiation of melanocytes between a/a and A/A mice . However, the content of pheomelanin in culture media from A/A melanocytes was increased by L-tyrosine compared with a/a melanocytes . In addition, the content of the pheomelanin precursor, 5-S-cysteinyldopa, in culture media from A/A melanocytes was dramatically increased by L-tyrosine . Moreover, pheomelanin content in the epidermis from 3.5- and 5.5-d-old A/A mice was much higher than in a/a mice . Analysis of the A gene using reverse transcription-polymerase chain reaction revealed that cultured keratinocytes and melanocytes do not express the A gene . Moreover, the A gene was expressed in the A/A dermis of 0.5-, 3.5- and 5.5-d-old mice, but not in the a/a dermis nor in the A/A or a/a epidermis . These results suggest that A/A epidermal melanoblasts are influenced by the A gene from the dermis of neonatal mice, and are capable of synthesizing pheomelanin in the culture . Pheomelanin production in the epidermis from 3.5- and 5.5-d-old A/A mice may be induced by the expression of the agouti gene in the dermis.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2004 Sep, 98(3), 301 - 10
Oropharyngeal candidiasis in HIV-infected patients under treatment with protease inhibitors; Migliorati CA et al.; OBJECTIVE: Oropharyngeal candidiasis decreased when protease inhibitors were included with other antiretrovirals to treat HIV infection . We tested oral yeast isolates of Brazilian HIV-infected individuals receiving antiretroviral therapy for protease secretion and susceptibility to ritonavir and some antifungals . STUDY DESIGN: We collected oral samples and identified yeasts from 19 HIV-infected patients receiving highly active antiretroviral therapy (HAART) and suspected of having oral candidiasis . Ritonavir and its excipients' effects on the isolated yeasts were tested for protease secretion by Ruchel's technique . The yeasts' susceptibility to amphotericin B (AnB), fluorocitosine (5FC), fluconazole (FZL), ketoconazole (KZL), and itraconazole (IZL) was determined by E-test (AB Biodisk) . Chi-squared test determined the statistical differences . RESULTS: Twenty-five different positive isolates were obtained . Sixty-eight percent were C . albicans . Other isolates included C . famata (16%), C . glabrata (4%), C . tropicalis (4%), T . capitatum (4%), and 1 isolate not identified . High protease secretion was observed for most of the isolates (20/25) . Ritonavir only altered enzyme secretion in 6/20 of the protease-secreting isolates . All isolates were highly sensitive to both AnB and 5FC . Antifungal activity did not change when ritonavir was added to the culture media . Some isolates were highly resistant to studied antifungals (52.2% KZL, 30.4% FZL, and 26% IZL) . Resistance significantly decreased when ritonavir was added to the medium with KZL and IZL (P <.5 by chi-squared) . A trend to decreased resistance was also observed with FZL but the results were not statistically significant . CONCLUSION: Candida continues to be the most prevalent fungus in the oral cavity . Although oral candidal isolates secrete protease, ritonavir does not inhibit all protease-secreting oral yeast isolates . There seems to be a synergistic effect between ritonavir and oral antifungals against fungal resistance.

Am J Physiol Renal Physiol, 2005 Jan, 288(1), F125 - 32 Epub 2005 Jan.
Stress response inhibits the nephrotoxicity of cisplatin; Hanigan MH et al.; Salt loading and saline hydration are used to protect patients from cisplatin-induced nephrotoxicity . The mechanism by which salt exerts its protective effect is unknown . As part of an ongoing study of cisplatin nephrotoxicity, an in vitro assay system was developed that models the in vivo exposure and response of proximal tubule cells to cisplatin . In this study, it was discovered that the toxicity of cisplatin toward LLC-PK(1) cells varied dramatically according to the tissue culture media used for 3-h cisplatin exposure . Further experiments revealed that minor variations in the sodium concentration among standard tissue culture media modulated cisplatin nephrotoxicity . NaCl has been shown to protect against cisplatin-induced nephrotoxicity in vivo but has never before been demonstrated in vitro . NaCl did not alter the cellular accumulation of cisplatin . NaCl altered the osmolarity of the external media, and its effect was replicated by substituting equiosmolar concentrations of impermeant anions or cations . The change in osmolarity triggered a stress response within the cell that modulated sensitivity to cisplatin . These data resolve several long-standing controversies regarding the mechanism by which salt loading protects the kidney from cisplatin-induced nephrotoxicity.

J Altern Complement Med, 2004 Aug, 10(4), 687 - 91
Inhibition of prostate cancer-cell proliferation by Essiac; Ottenweller J et al.; OBJECTIVE: To assess the ability of Essiac tea extracts (Essiac Canada International, Ottawa, Canada) to modulate cancer cell proliferation and immune responsiveness . DESIGN: A noncancerous transformed cell line was compared to a cancerous cell line and spleen cells that had been isolated from mice to examine proliferation responses mediated by the addition of an Essiac preparation . RESULTS: We found in vitro evidence of decreased proliferation of both noncancerous transformed (CHO) and cancerous prostate cell line (LNCaP) when Essiac was present in the culture media . A dose response for inhibition was demonstrated by a linear regression performed on the data for both the CHO and LNCaP cells . The percent inhibition of the LNCaP cells was higher than the percent inhibition of the CHO cells suggesting that Essiac may have a more selective effect on cancer cells than transformed cells . In addition, the effects of Essiac were examined in an immune T-lymphocyte proliferation assay . At low doses of Essiac, augmentation of proliferation of these T cells was demonstrated, but at higher doses Essiac was inhibitory to T-cell proliferation . The same doses of Essiac that stimulated spleen cells were inhibitory for LNCaP cell proliferation . CONCLUSIONS: Essiac preparations may be able to inhibit tumor cell growth while enhancing immune response to antigenic stimulation . This may be especially valuable in immune-suppressed individuals.

Org Biomol Chem, 2004 Sep 21, 2(18), 2578 - 84 Epub 2004 Aug 20.
Reactivity of hydrazinophthalazine drugs with the lipid peroxidation products acrolein and crotonaldehyde; Kaminskas LM et al.; The nucleophilic drug hydralazine strongly inhibits cell toxicity mediated by acrolein, a short chain 2-alkenal formed during lipid peroxidation . We here report the chemistry of acrolein-trapping by hydralazine, and show that together with its structural analogue dihydralazine, it also readily traps crotonaldehyde . Isolable reaction products included (1E)-acrylaldehyde phthalazin-1-ylhydrazone (E-APH), (1Z)-acrylaldehyde phthalazin-1-ylhydrazone (Z-APH), (1E,2E)-but-2-enal phthalazin-1-ylhydrazone (E-BPH) and (1Z,2E)-but-2-enal phthalazin-1-ylhydrazone (Z-BPH) . Concentration-dependent formation of (1E)-acrylaldehyde phthalazin-1-ylhydrazone was observed in the culture media of cells co-exposed to hydralazine and the acrolein precursor allyl alcohol . These aldehyde-sequestering properties of hydrazinophthalazine drugs may contribute to the protection they provide against 2-alkenal-mediated toxicity.

J Mater Sci Mater Med, 1998 Dec, 9(12), 815 - 8
Interactions of chondrocytes with methacrylate copolymers; Hutcheon GA et al.; Copolymers of poly(ethylmethacrylate) (PEMA) and tetrahydrofurfurylmethacrylate (THFMA) have been shown to exhibit potential as a biomaterial for use in cartilage repair . However, the interactions of chondrocytes with the polymer surface is not well understood . A series of novel methacrylate copolymers containing PEMA, THFMA and hydroxyethylmethacrylate (HEMA) were prepared and the ability of these various copolymers to support chondrocytes attachment in vitro has been assessed by the Alamar blue assay for cell number and environmental scanning electron microscopy (ESEM) . As the mole fraction of HEMA in PEMA/THFMA/HEMA copolymers increased, chondrocyte attachment to the polymer surface in 24 h decreased . Chondrocytes maintained a rounded morphology and were strongly attached on the THFMA/PEMA polymer surface, but as the mole fraction of HEMA increased the cells present became much smaller with fewer cell to cell interactions . The effect of pre-adsorbing fibronectin on to the polymer surface on cell attachment was assessed both in the presence and absence of serum . Chondrocyte attachment was significantly reduced in serum-free medium . Pre-adsorption of fibronectin on to the copolymer surface substantially increased cell attachment in all cases . In conclusion, chondrocyte attachment and proliferation on these copolymers may be controlled by changes in the polymer surface chemistry and is highly sensitive to the presence of proteins either in the culture media or pre-adsorbed on to the copolymer surface .

J Mater Sci Mater Med, 2001 Aug, 12(8), 693 - 8
Biochemical and histological evaluation of human synovial-like membrane around failed total hip replacement prostheses during in vitro mechanical loading; Bosetti M et al.; The biochemical role of the synovial-like membrane formed at the interface of eight aseptic failed total hip prosthesis has been investigated during in vitro mechanical loading . The study was carried out on four membranes from cemented prosthesis and four titanium alloy uncemented ones . Intermittent positive pressure leading to 20% deformation of the membrane (100 g/cm(2))was applied to the membrane fragments in cycles (300 cycles/15 min) repeated three times at thirty minutes intervals in which interleukin-6 (IL6), prostaglandin-E2 (PGE2) and interleukin-1beta (IL1beta) levels were quantified both in culture media and in tissue extracts . Histological, morphometrical and immunohistochemical studies were also carried out on the same membranes . Mechanical stress evidenced an increase in the release of the examined cytokines both in cemented and uncemented prosthesis tissues; particularly evident was IL6 trend of increase from cemented prosthesis and IL1beta result from uncemented ones . Histomorphological and immunohistochemical data revealed no differences between membranes obtained from cemented and uncemented prosthesis as to cell proliferation, fibrosis, macrophages lymphocytes B and T population, vessels and nervous fibers . The results indicate that mechanical stress plays a fundamental role in increasing membrane production and release of cytokines known as bone-resorbing agents . Furthermore, the histologic finding of synovial-like membrane with the same histomorphological and immunohistochemical findings but with different biochemical response to mechanical stimulation, suggests that cells involved in the production and release of the considered mediators might have different strain behavior by different development conditions (previous contact with PMMA) .

J Mater Sci Mater Med, 2001 Sep, 12(9), 833 - 44
The influence of crosslinking agents and diamines on the pore size, morphology and the biological stability of collagen sponges and their effect on cell penetration through the sponge matrix; McKegney M et al.; Artificial skin substitutes based on autologous keratinocytes cultured on collagen substrata are being developed for treating patients with severe burns . The properties of the collagen substrate can be manipulated, for example, by crosslinking, to optimize desirable properties such as cell growth and penetration into the substrate, biological stability and mechanical strength . Collagen sponges crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and the diamine, diaminohexane, were used to determine the effect of crosslinking on pore size and morphology, on the stability of the crosslinked sponges both in cell culture media and during incubation with collagenase, and on the penetration of keratinocytes and fibroblasts through the sponge matrix . Crosslinking of the sponges reduced the pore size, particularly at the surface, and altered sponge morphology . After crosslinking the collagen fibers were thinner, and appeared lacy and delicate . Crosslinking also influenced sponge stability . In keratinocyte serum-free medium the pore size of plain collagen sponges increased with increasing incubation time, and crosslinking appeared to prevent this, and may have stabilized sponge structure . Incubation in serum-containing Dulbecco's minimum essential medium caused a marked reduction in pore size in both plain collagen and crosslinked collagen sponges . Crosslinking did not appear to influence this cell-free contraction of collagen sponges . Treatment of sponges with EDAC markedly increased the resistance of sponges to collagenase digestion . The penetration of both keratinocytes and fibroblasts was retarded by crosslinking the sponges . Fibroblasts penetrated through the sponges to a greater extent than keratinocytes, and their proliferation rate was faster . The total number of cells populating the crosslinked sponges after 10 days culture was approximately 50% of that on untreated collagen sponges . The mechanism responsible for this effect was different with the two crosslinkers used . Diaminohexane appeared to inhibit cell growth, whereas EDAC may have caused a decrease in cell adhesion to the sponges, without an apparent inhibition of growth rate . In terms of morphology, fibroblasts were elongated to a greater extent on crosslinked sponges, and alligned themselves along the collagen fibers . Keratinocytes grew in colonies on untreated sponges, but on crosslinked sponges they grew in isolation, with minimal cell-cell interactions . It may be necessary to reach a compromise to obtain the best combination of properties for using collagen sponges as substrata for artificial skin substitutes .

J Mater Sci Mater Med, 1999 Sep, 10(9), 567 - 76
In vitro growth and differentiation of osteoblast-like human bone marrow cells on glass reinforced hydroxyapatite plasma-sprayed coatings; Ferraz MP et al.; Human osteoblastic bone marrow cells were cultured for periods of up to 28 days in control conditions and on the surface of a glass reinforced hydroxyapatite composite (HA/G1) and commercial hydroxyapatite (HA) plasma-sprayed coatings, in the "as-received" condition and after immersion treatment in culture medium for 21 days . Cultures were characterized for total protein content and alkaline phosphatase activity . Scanning electron microscope analyses were performed on control cultures, seeded materials and materials incubated in the absence of cells . Culture media were analyzed for total and ionized calcium and phosphorus concentrations throughout the incubation period . Immersion of HA/G1 and HA coatings in culture medium resulted in significant alterations to the levels of calcium and phosphorus in the medium, leading to surface modifications . However, seeded material samples showed significant differences in the pattern of variation of the levels of these species . Cell proliferation was observed in the "as-received" HA/G1 composite, but cell mediated formation of mineral deposits was not proved . In contrast, "as-received" HA hardly supported cell growth . Previously immersed material samples showed cell proliferation and evidence of biological formation of mineral deposits . However, the HA/G1 composite presented better surface characteristics for cell growth as the behavior of bone marrow cells was closer to that observed in control cultures .

J Mater Sci Mater Med, 1999 Dec, 10(12), 761 - 5
Image and fractal analysis of osteoblastic cells in viscous media; Pearson M et al.; The aim of the present study was to determine whether osteoblastic morphology was affected by the viscosity of culture media and whether any morphological differences could be readily quantified . A cytochemical stain was used for alkaline phosphatase and a combination of image and fractal analysis, utilising seven morphological parameters to assess the morphology of osteoblasts . Using regression analysis it was determined that as the viscosity of the culture media increased the area of the cells significantly decreased and the fractal dimension of the cell profiles significantly increased . A discriminant function analysis was used to examine whether cell populations could be classified according to culturing time or the viscosity of culturing media based on the seven morphological parameters . It was determined that the cells could be classified up to 93% correct according to the viscosity of the media they were cultured in and up to 93.5% correct according to the culturing time . This study demonstrated that viscous media affects the morphology of osteoblastic cells and that discriminant function analysis can be used to classify these cells based on their morphological parameters .

J Biochem Biophys Methods, 2004 Sep 30, 60(3), 191 - 203
Analysis of amino acid-carbohydrate mixtures by anion exchange chromatography and integrated pulsed amperometric detection; Jandik P et al.; A review is presented of recent developments in the area of analysis of amino acid-carbohydrate mixtures . Based on its broad selectivity, the AminoPac PA10 column exhibits a remarkable capability to perform simultaneous separations of amino acids and carbohydrates . This ability is further enhanced by the equal sensitivity for carbohydrates and amino acids exhibited by the "amino acid" integrated pulsed electrochemical detection (IPAD) waveforms . Equimolar levels of carbohydrates and amino acids are separated either by optimized elution gradients alone or by a combination of modified gradients and a bi-modal IPAD waveform . Samples containing large amounts of carbohydrates may be analyzed after suitable sample preparation . Both a manual off-line method and a fully automatic on-line method are discussed . In addition, we will review the application of these methods to various types of samples, including cell culture media, glycoprotein hydrolysates, beverages, condiments and soil extracts.

Stem Cells Dev, 2004 Aug, 13(4), 325 - 36
Culture and characterization of human embryonic stem cells; Draper JS et al.; Human embryonic stem (ES) cells offer substantial opportunities for providing well-defined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for their large-scale culture under defined conditions, and for controlling and directing their differentiation, presents a substantial challenge . Markers for defining the undifferentiated cells are well established, based upon previous studies of embryonal carcinoma (EC) cells, their malignant counterparts from teratocarcinomas . These provide valuable tools for monitoring human ES cultures and their state of differentiation . However, current culture techniques are suboptimal and involve the use of poorly defined culture media and the use of feeder cells . Over time, the cells may also acquire karyotypic changes, reflecting genetic selection and adaptation to in vitro culture conditions . Nevertheless, progress is being made . Originally, human ES cells were derived and maintained in medium containing fetal calf serum . They are now widely cultured in a proprietary serum-free formulation (serum replacement from Invitrogen Corp., Carlsbad, CA), and recently we have derived a new human ES line in this medium without fetal calf serum . Human fibroblasts can also be used to replace mouse embryo fibroblasts as feeder cells . We have now found it possible to culture a subline of human ES cells on Matrigel, or purified collagen type IV, laminin, and fibronectin, without feeders or feeder-conditioned medium . These cells nevertheless retain the features of undifferentiated human ES cells, including a capacity for differentiation . Although these cells also carried karyotypic changes, further research focused upon understanding the mechanisms that control self-renewal, apoptosis, and commitment to differentiation will facilitate the development of defined culture conditions that minimize genetic change and optimize the maintenance of the undifferentiated stem cells.

Bone, 2004 Sep, 35(3), 689 - 96
Family 2 cystatins inhibit osteoclast-mediated bone resorption in calvarial bone explants; Brand HS et al.; Osteoclastic bone resorption depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases . Biochemical studies have shown that cystatins, naturally occurring inhibitors of these enzymes, inhibit bone matrix degradation . Since the mechanism by which cystatins exert this inhibitory effect is not completely resolved yet, we studied the effect of cystatins on bone resorption microscopically and by Ca-release measurements . Calvarial bone explants were cultured in the presence or absence of family 2 cystatins and processed for light and electron microscopic analysis, and the culture media were analyzed for calcium release . Both egg white cystatin and human cystatin C decreased calcium release into the medium significantly . Microscopic analyses of the bone explants demonstrated that in the presence of either inhibitor, a high percentage of osteoclasts was associated with demineralized non-degraded bone matrix . Following a 24-h incubation in the presence of cystatin C, 41% of the cells were adjacent to areas of demineralized non-degraded bone matrix, whereas in controls, this was only 6% . If bone explants were cultured with both PTH and cystatin C, 60% of the osteoclasts were associated with demineralized non-degraded bone matrix, compared to 27% for bones treated with PTH only (P < 0.01) . Our study provides evidence that cystatins, the naturally occurring inhibitors of cysteine proteinases, reversibly inhibit bone matrix degradation in the resorption lacunae adjacent to osteoclasts . These findings suggest the involvement of cystatins in the modulation of osteoclastic bone degradation.

J Neurosci Res, 2004 Sep 15, 77(6), 892 - 900
Protective effect of green tea polyphenol EGCG against neuronal damage and brain edema after unilateral cerebral ischemia in gerbils; Lee H et al.; Previous studies have demonstrated that a green tea polyphenol, (-)-epigallocatechine gallate (EGCG), has a potent free radical scavenging and antioxidant effect . Glutamate leads to excitotoxicity and oxidative stress, which are important pathophysiologic responses to cerebral ischemia resulting in brain edema and neuronal damage . We investigated the effect of EGCG on excitotoxic neuronal damage in a culture system and the effect on brain edema formation and lesion after unilateral cerebral ischemia in gerbils . In vitro, excitotoxicity was induced by 24-hr incubation with N-methyl-D-aspartate (NMDA; 10 microM), AMPA (10 microM), or kainate (20 microM) . EGCG (5 microM) was added to the culture media alone or with excitotoxins . We examined malondialdehyde (MDA) level and neuronal viability to evaluate the effect of EGCG . In vivo, unilateral cerebral ischemia was induced by occlusion of the right common carotid artery for 30, 60, or 90 min and followed by reperfusion of 24 hr . Brain edema, MDA, and infarction were examined to evaluate the protective effect of EGCG . EGCG (25 or 50 mg/kg, intraperitoneally) was administered twice, at 30 min before and immediately after ischemia . EGCG reduced excitotoxin-induced MDA production and neuronal damage in the culture system . In the in vivo study, treatment of gerbils with the lower EGCG dose failed to show neuroprotective effects; however, the higher EGCG dose attenuated the increase in MDA level caused by cerebral ischemia . EGCG also reduced the formation of postischemic brain edema and infarct volume . These results demonstrate EGCG may have future possibilities as a neuroprotective agent against excitotoxicity-related neurologic disorders such as brain ischemia.

Reproduction, 2004 Sep, 128(3), 281 - 91
Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development; Jurisicova A et al.; Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under current in vitro culture conditions . There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions . In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality . An increasing body of evidence indicates that cell fate (i.e . survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development . The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development . In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.

Biol Trace Elem Res, 2004 Aug, 100(2), 151 - 68
Metallic dental material biocompatibility in osteoblastlike cells: correlation with metal ion release; Cortizo MC et al.; Ions released from metallic dental materials used in orthodontic appliances could induce undesirable effects on cells and tissues . This study evaluates the biocompatibility of two of the most labile components of metallic dental alloys on osteoblastlike cells . The influence of protein and ions on metal dissolution properties is also investigated using different electrolyte solutions . Morphological alterations, cell growth, and differentiation of osteoblasts were assessed after exposure to pure metals (Ag, Cu, Pd, Au) and Ni-Ti alloy and correlated with the kinetics of elements released into the culture media . Results showed that Cu and Ag were the most cytotoxic elements and the other metals were biocompatible with the osteoblasts . The parameters of biocompatibility were correlated with the levels of ions detected into the culture media . Metal ions induced cell death through early mitosis arrest, apoptotic phenomena, and necrotic processes . Voltammograms showed that anions and proteins interfered in the corrosion process . Fetal bovine serum (FBS) strongly affected the electrochemical process, decreasing the oxidation rate of the metals . In conclusion, copper and silver ions showed a time-dependent low biocompatibility, which correlated with the concentration of released ions . The dissolution of the metallic materials was dependent on the composition of the simulated biological media.

Alcohol Clin Exp Res, 2004 Aug, 28(8 Suppl Proceedings), 145S - 147S
The phosphodiesterase III inhibitor olprinone decreases sensitivity of rat Kupffer cells to endotoxin; Enomoto N et al.; BACKGROUND: Sensitivity of Kupffer cells to endotoxin {lipopolysaccharide (LPS)} and overproduction of tumor necrosis factor-alpha (TNF-alpha) are critical for progression of alcoholic liver injury . Therefore, suppression of TNF-alpha should prove useful for treatment of alcoholic liver injury . However, a transient increase of intracellular calcium ({Ca}i) is required for LPS-induced TNF-alpha production by the macrophage cell line . The phosphodiesterase III inhibitor olprinone has been shown to suppress {Ca}i level in vascular smooth muscle cells . Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin . METHODS: Kupffer cells were isolated by collagenase digestion and differential centrifugation . LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 micromol/liter) . After addition of LPS (10 microg/ml) to culture media, {Ca}i was measured using a fluorescent indicator, fura-2 . RESULTS: LPS increased {Ca}i of Kupffer cells in control rats from basal levels (28 +/- 4 nmol/liter) to 280 +/- 14 nmol/liter . This increase was blunted by olprinone (91 +/- 8 nmol/liter) . Similarly, olprinone diminished the LPS (1 microg/ml)-induced TNF-alpha production by Kupffer cells by 30% (2220 +/- 116 vs . 1386 +/- 199 pg/ml; p < 0.05) . CONCLUSIONS: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin.

Respiration, 2004 Jul-Aug, 71(4), 360 - 6
Isolation of fungi, especially Exophiala dermatitidis, in patients suffering from cystic fibrosis . A prospective study; Horre R et al.; BACKGROUND: Patients with cystic fibrosis (CF) are at an increased risk of pulmonary colonisation by opportunistic micro-organisms . Using specialised methods, the black yeast Exophiala dermatitidis could consistently be cultured from CF patients . Isolation rates from sputum samples ranged between 1.8 and 15.7% . Occasionally, infection could be recognised . OBJECTIVES: This study aimed at investigating the isolation rates of E . dermatitidis in samples taken from CF patients at the University of Bonn, Germany . METHODS: Altogether, 439 respiratory specimens taken from 81 CF patients were screened for the occurrence of E . dermatitidis over a period of 18 months . For the selective isolation of this fungus erythritol-chloramphenicol agar (ECA) produced in house was applied . RESULTS: The isolation rate of E . dermatitidis was 1.1% from all specimens, 1.6% from all sputum samples and 6.2% in all patients examined . CONCLUSIONS: Prior to the introduction of ECA, E . dermatitidis had never been isolated in our laboratory, either from CF, or from any other patient . During this study, E . dermatitidis was found to colonise the respiratory tract of some CF patients . The use of additional selective culture media is necessary for the recognition of uncommon fungi, e.g . E . dermatitidis, in CF patients .

Biochim Biophys Acta, 2004 Aug 23, 1693(2), 111 - 23
Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A; Kundranda MN et al.; Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media . We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion . Interestingly, the cells can also divide and attain confluency under these conditions . Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions . Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes . Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion . Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion . Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.

J Anim Sci, 2004 Jul, 82(7), 1967 - 75
Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro; Mao J et al.; The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis . Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp . 1) or alpha-minimum Eagle's essential medium (Exp . 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp . 1 and 3) or absence (Exp . 2) of 7.5% fetal calf serum . According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp . 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp . 2 and 3) were added to the culture media . In Exp . 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose . In Exp . 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone . In Exp . 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I . Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation . These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.

Mol Microbiol, 2004 Aug, 53(4), 1109 - 21
Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli; Fraile S et al.; The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined . A strong dimerization domain (i.e . an amphipathic alpha-helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope-tagged version of the 23 kDa C-terminal secretion signal of haemolysin (EHlyA) . The zipper-containing polypeptide (ZEHlyA) was effectively secreted by E . coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography . In vivo protein cross-linking experiments and coexpression with a secretion-deficient derivative of ZEHlyA indicated that leucine zipper-dependent dimerization occurs following secretion . To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin V(HH)-domains obtained from camel antibodies were fused to EHlyA and ZEHlyA . Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e . avidity) of the V(HH) moiety, thus becoming an excellent reporter of correct protein folding and dimerization . Both V(HH)-EHlyA and V(HH)-ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively . When compared with their monomeric counterparts, the dimeric V(HH)-ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10-fold increase in their avidity . These data reveal a non-anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity.

J Orthop Res, 2004 Sep, 22(5), 1135 - 42
The use of a non-ionic surfactant (P188) to save chondrocytes from necrosis following impact loading of chondral explants; Phillips DM et al.; While current injury criteria for the automotive industry are based on bone fracture, the majority of knee injuries suffered in collisions each year do not involve fracture of bone . Instead, clinical studies of traumatic joint injury often document early pain and development of chronic diseases, such as osteoarthritis . Previous studies suggest chronic disease can be initiated by cell death that occurs in articular cartilage during mechanical trauma to the joint . In the current investigation early necrosis of chondrocytes was investigated after blunt trauma to chondral explants . A non-ionic surfactant (P188) was explored as a potential tool for early intervention into the disease process, as this surfactant has been shown to repair damaged membranes in other cell lines . Three groups of adult bovine chondral explants were equilibrated for 48 h in culture media . Two groups were then loaded to 25 MPa in unconfined compression . Half the specimens in each group were incubated in media supplemented with 8 mg/ml P188 immediately after loading, while the other half was returned to standard media . At 1 and 24 h the percentages of live and dead cells in compressed and control groups were determined with a cell viability stain . At 1 h post-trauma, P188 incubated specimens had a significantly increased percentage of live cells in the superficial zone versus the no-P188 group . At 24 h the percentages of live cells in all three zones of the P188-treated explants were significantly greater than in the no treatment group . This study showed that P188 surfactant could help restore the integrity of cell membranes in cartilage damaged by blunt mechanical trauma . With the ability of P188 to "save" chondrocytes from early necrotic death using in vitro chondral explants, its role in prevention of a post-traumatic osteoarthritis in a diarthrodial joint should be further explored using in vivo animal models.

Folia Biol (Krakow), 2003, 51(3-4), 181 - 8
Hormonal regulation of oestrogen formation by Leydig cells in vitro: immunocytochemical approach; Gancarczyk M et al.; The ability of the testis to convert androgens into oestrogens is related to the presence of a microsomal enzyme, aromatase, in testicular cells . The aim of this study was to show whether the supplementation of culture media with LH or an aromatase inhibitor could affect the process of aromatisation in Leydig cells of the bank vole in vitro . This was investigated by means of immunocytochemistry and radioimmunological assays . In control cultures of Leydig cells, both steroid hormones secretion as well as immunoreactivities for aromatase and oestrogen receptor were weaker than in those treated with LH . On the contrary, the addition of aromatase inhibitor into the culture medium resulted in a decreased intensity of immunocytochemical stainings in comparison with the control . Concomitantly, the androgen level was slightly higher, whereas that of oestrogen significantly lower than in the control cultures . Additionally, to check whether steroid hormones are able to regulate aromatase or oestrogen receptor immunoexpressions, some of the Leydig cell cultures were enriched with testosterone or oestradiol, respectively . Strong immunoreactivities for both aromatase and oestrogen receptor were observed . This suggests that Leydig cells in vitro are able to regulate directly the secretion of oestrogens by active aromatase . Finally, it is concluded that oestrogen formation in bank vole Leydig cells in vitro can be influenced by various factors . It should be stressed, however, that the effect of hormone stimulation or aromatase inhibitor action appeared to be dependent on the length of light cycles that bank voles were exposed prior to the isolation of Leydig cells.

Microbiol Res, 2004, 159(2), 147 - 56
Optimization of expression of an annexin V-hirudin chimeric protein in Escherichia coli; Yuan H et al.; A human Annexin V-Hirudin chimeric protein, Annexin V-Hirudin C, was expressed in Escherichia coli . A broad range of parameters such as plasmid stability during propogation and expression, expression capacity stability, the culture media, the growth time before induction and the induction duration were examined and optimized . Recombinant Annexin V-Hirudin C was purified from the cell lysate supernatants by ethanol precipitation, DEAE-cellulose chromatography and Sephadex G-75 chromatography, and the purified protein showed dose-dependent thrombin inhibitory activity . The overall production of purified Annexin V-Hirudin C protein is 10 mg/l/OD600.

Mikrobiyol Bul, 2004 Jan-Apr, 38(1-2), 51 - 9
{Investigation of Herpes simplex virus type 1 in the samples of patients with clinically prediagnosed as herpetic keratitis or keratoconjunctivitis}; Biriken D et al.; The aim of this study was to investigate the presence of Herpes simplex virus type 1, in the specimens from patients clinically prediagnosed as herpetic keratitis or keratoconjunctivitis, by virus isolation and direct immunoperoxidase methods . The samples obtained from a total of 33 patients included ulcerated corneal epithelium scrapings and, swabs from ulcer, corneal epithelium, and lower conjunctivas . For virus isolation, both scraping and swab samples for each patient, were inoculated into monolayered Vero cell lines which have previously cultivated on 24-well plates, and examined for the presence of cytopathic effects typical for HSV-1 . At the end of 5-days incubation period, the culture media were discarded, the cells were fixed and stained with peroxidase labeled specific HSV-1 antibodies (direct immunoperoxidase--DIP--method) . Simultaneously, the smears were prepared from the samples which were sufficient in amount, and stained with DIP method . As a result, cytopathic effects on cell cultures were observed in 4 (12.1%) of the samples, of which 2 were also positive with DIP method . Following DIP staining of smears prepared from 15 samples, HSV-1 antigen positivity was detected in only 1 of the samples . This sample was negative in cell culture . In contrast, one of the 2 cell culture positive samples yielded negative result in smear examination, while the other could not be examined since the sample was not enough . In conclusion, HSV-1 has been shown as the etiologic agent in 3 (9.1%) of our patients clinically prediagnosed as herpetic keratitis or keratoconjunctivitis, and lesion scrapings were determined as the most appropriate samples for virus isolation.

Oecologia . 2004 Aug 3; {Epub ahead of print}
Filamentous cyanobacteria, temperature and Daphnia growth: the role of fluid mechanics; Abrusan G; Viscosity increases significantly with a fall in water temperature, thus temperature change affects not only the metabolic rates of aquatic suspension feeders, but also the physical properties of the surrounding fluid . This mechanistic effect of water temperature change on growth was separated from the effect of metabolism by using culture media with modified viscosity, while the temperature was kept constant . The effect of water viscosity on growth rate and feeding of four Daphnia species ( D . magna, D . pulicaria, D . hyalina, D . galeata) was investigated . Increased viscosity decreased the growth rate significantly for three species, with the exception of D . galeata . Changing viscosity also affects growth qualitatively: the filamentous blue-green Cylindrospermopsis raciborskii reduces the growth rate of D . pulicaria at low viscosity, but its negative effect disappears when viscosity is higher . The findings are consistent with the hypothesis that it is the Reynolds number of the filtering appendages that determines the qualitative features of Daphnia filtration . The edibility of C . raciborskii at high water viscosity is most probably caused by lack of interference with filtering combs, and explains the coexistence of D . pulicaria with filamentous blue-green species in the field, and also the observed temperature dependence of growth inhibition of filaments.

Plant Cell Rep . 2004 Jul 28; {Epub ahead of print}
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.

Curr Eye Res, 2004 May, 28(5), 327 - 36
Alternative culture conditions for isolation and expansion of retinal progenitor cells; Qiu G et al.; PURPOSE: To investigate different in vitro model systems for retinal progenitor cell (RPC) isolation and expansion . METHODS: RPCs were isolated from embryonic day (E) 17 Long Evans rat retinas . Three different culture media: (1) modified serum free defined media (2) serum-containing media and (3) embryonic stem cell (ES)-conditioned media were used for RPC isolation and long term expansion . Expression of various cellular markers and cell morphologies were compared among the three culture systems at different passages by immunostaining and confocal microscopy . RESULTS: All three culture systems could maintain RPCs as nestin-positive cells (78-87%) after long-term in vitro expansion . However, RPCs appeared to proliferate faster in the serum-free culture system . The ES-conditioned media provided the best RPC survival . Cells appeared smaller at early passages compared with later passages . This morphology change occurred at P9-P10 in the serum-free medium, and at P5-P6 in the other two culture systems . CONCLUSIONS: The serum-free medium may be superior for preventing RPC differentiation during expansion.

Poult Sci, 2004 Jul, 83(7), 1193 - 8
Proliferative and steroidogenic effects of follicle-stimulating hormone on cultured chick embryo testis cells; Peralta I et al.; The present study evaluated the follicle-stimulating hormone (FSH) effect on cell proliferation and steroid production by chick embryo testis . Dissociated cells from 18-d-old embryos were cultured on polycarbonate membranes in defined media . In some experiments, cells were further separated by a metrizamide gradient, and 5 cellular subpopulations were recovered and cultured . {3H}thymidine was added to the culture media . When necessary, 17beta-estradiol, human FSH (hFSH), recombinant human FSH (rhFSH), or human chorionic gonadotropin (hCG) was added to the medium at the beginning of the culture . The total number of cells and the incorporation of {3H}thymidine increased when hFSH or rhFSH was added . No changes were produced by the addition of hCG or 17beta-estradiol . The dose-response curve to hFSH resulted in an ED50 of 0.25 IU/mL . The stimulatory effect of hFSH on total number of cells and on {3H}thymidine incorporation was significant at 36 h of culture and was maintained up to 60 h . Testosterone production increased with the addition of FSH or rhFSH, meanwhile estradiol production was below the limit of detection of RIA . The hFSH proliferative effect measured as {3H}thymidine incorporation was observed only in the F3, F4, and F5 fractions of the density gradient . Present results show that hFSH and rhFSH, but not hCG or estradiol, stimulate testis cell proliferation in a time- and dose-dependent manner . The combination of {3H}thymidine incorporation and testosterone production in fractions obtained from the metrizamide density gradients suggests that the cell fractions of the chick embryo testis show a differential response to FSH.

Am J Obstet Gynecol, 2004 Jun, 190(6), 1557 - 62; discussion 1562-3
Racial disparity in membrane response to infectious stimuli: a possible explanation for observed differences in the incidence of prematurity . Community Award Paper; Fortunato SJ et al.; OBJECTIVE: This study compares the immune responsiveness of amniochorionic membranes (AC) derived from African American (AA) and white (C) women to an infectious stimulus ex vivo . STUDY DESIGN: AC derived from AA and C women were placed in an organ explant culture for 48 hours and then stimulated with endotoxin . Enzyme-linked immunosorbent assay measured the concentration of matrix metalloproteinase 9 (MMP9), tumor necrosis factor-alpha (TNF-alpha), and soluble TNF receptors (sTNFR1 and sTNFR2) in culture media from stimulated and unstimulated AC . RESULTS: The C group produced 8-fold more TNF-alpha after stimulation than did the AA group . Both soluble receptor (R1 and R2) production increased in the C group and decreased in the AA group after stimulation . Although the C group-derived membranes produced more MMP9 at rest, a 6-fold increase in MMP9 concentration was seen in the AA group-derived membranes after stimulation . No change in MMP9 concentration was seen after stimulation of the C group-derived membranes . CONCLUSION: Although the C group produced more TNF, they also produce higher sTNFRs, which may serve a protective role . The increased MMP9 release by the AA group may be suggestive of the greater risk of premature rupture of membranes in the AA group.

J Biol Chem, 2004 Oct 8, 279(41), 43227 - 36 Epub 2004 Jul 28.
Caspase-3-induced truncation of type 1 inositol trisphosphate receptor accelerates apoptotic cell death and induces inositol trisphosphate-independent calcium release during apoptosis; Assefa Z et al.; Inositol 1,4,5-trisphosphate receptor-deficient (IP3RKO) B-lymphocytes were used to investigate the functional relevance of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) and its cleavage by caspase-3 in apoptosis . We showed that inositol 1,4,5-trisphosphate receptor-deficient cells were largely resistant to apoptosis induced by both staurosporine (STS) and B-cell receptor (BCR) stimulation . Expression of either the wild-type IP3R1 or an N-terminal deletion mutant (Delta1-225) that lacks inositol 1,4,5-trisphosphate-induced Ca2+ release activity restored sensitivity to apoptosis and the consequent rise in free cytosolic Ca2+ concentration ({Ca2+}i) . Expression of caspase-3-non-cleavable mutant receptor, however, dramatically slowed down the rate of apoptosis and prevented both Ca2+ overload and secondary necrosis . Conversely, expression of the "channel-only" domain of IP3R1, a fragment of the receptor generated by caspase-3 cleavage, strongly increased the propensity of the cells to undergo apoptosis . In agreement with these observations, caspase inhibitors impeded apoptosis and the associated rise in {Ca2+}i . Both the staurosporine- and B-cell receptor-induced apoptosis and increase in {Ca2+}i could be induced in nominally Ca2+-free and serum-free culture media, suggesting that the apoptosis-related rise in {Ca2+}i was primarily because of the release from internal stores rather than of influx through the plasma membrane . Altogether, our results suggest that IP3R1 plays a pivotal role in apoptosis and that the increase in {Ca2+}i during apoptosis is mainly the consequence of IP3R1 cleavage by caspase-3 . These observations also indicate that expression of a functional IP3R1 per se is not enough to generate the significant levels of cytosolic Ca2+ needed for the rapid execution of apoptosis, but a prior activation of caspase-3 and the resulting truncation of the IP3R1 are required.

Endocrinology, 2004 Nov, 145(11), 4838 - 45 Epub 2004 Jul 29.
Cytotrophoblasts up-regulate soluble fms-like tyrosine kinase-1 expression under reduced oxygen: an implication for the placental vascular development and the pathophysiology of preeclampsia; Nagamatsu T et al.; Sufficient cytotrophoblast (CT) invasion into the uterine wall and subsequent remodeling of maternal uterine vasculature is critical to establish uteroplacental circulation . The production of vascular endothelial growth factor (VEGF) family molecules is confirmed in placental cells including CTs, but it is not elucidated how the VEGF system in CTs is controlled by oxygen tension and how it is involved in the development of placental circulation . To address this, we explored the effect of oxygen tension on the expression of VEGF, placenta growth factor (PlGF), and their antagonist, soluble fms-like tyrosine kinase-1 (sFlt-1) using ELISA and real-time PCR in a primary CT cell culture . For comparison, the same was conducted in parallel using other cells comprising placenta, such as human umbilical vein endothelial cells (HUVECs) and villous fibroblasts (VFs) . Reduced oxygen resulted in a pronounced increase in sFlt-1 mRNA amount and sFlt-1 release into the culture media in CTs, whereas this was not the case with HUVECs and VFs . Free (not bound to sFlt-1) VEGF was not detected in CT culture media regardless of oxygen concentration, even though VEGF expression was stimulated by reduced oxygen in CTs, which was similar to the stimulation in HUVECs and VFs . Free PlGF was also diminished in CT culture media by reduced oxygen . These results implicate that CTs possess a unique property to enhance sFlt-1 production under reduced oxygen, which could consequently antagonize angiogenic activity of VEGF and PlGF . The presented findings might provide a framework with which to understand the mechanism of uterine vascular remodeling and its perturbations as exemplified in preeclampsia.

J Pharmacol Exp Ther, 2004 Dec, 311(3), 1105 - 14 Epub 2004 Dec.
Inhibition of tumor cell proliferation by sigma ligands is associated with K+ Channel inhibition and p27kip1 accumulation; Renaudo A et al.; Previous studies have shown that sigma receptors are overexpressed in tumor cells . However, the role of sigma receptors remains enigmatic . Recently, we and others have demonstrated that sigma-1 receptor modulates K+ channels in pituitary . In the present report, patch-clamp and Western blot assays were used in small cell lung cancer (SCLC, NCI-H209, and NCI-H146) and leukemic (Jurkat) cell lines to investigate the effects of sigma ligands on voltage-gated K+ channels and cell proliferation . The sigma ligands (+)-pentazocine, igmesine, and 1,3-di(2-tolyl)guanidine (DTG) all reversibly inhibited voltage-activated K+ currents in both cell lines . The potency of sigma ligand-induced inhibition (10 microM) was igmesine = (+)-pentazocine > DTG, pointing to the involvement of sigma-1 receptors . Addition of the K+ channel blockers tetraethylammonium (TEA) and 4-aminopyridin or one of cited sigma ligands in the culture media reversibly inhibited Jurkat cell growth . Interestingly, K+ channel blockers and sigma ligands caused an accumulation of the cyclin-dependent kinase inhibitor p27kip1 and a decrease in cyclin A expression in Jurkat and SCLC cells, whereas no effect could be detected on p21cip1 . Moreover, sigma ligands and TEA had no effect on caspase 3 activity . Accordingly, incubation of cells with sigma ligands did not provoke DNA laddering . These data demonstrate that sigma ligands and voltage-dependent channel blockers inhibit cell growth through a cell cycle arrest in the G1 phase but not via an apoptotic mechanism . Altogether, these results indicate that the sigma-1 receptor-induced inhibition of the cell cycle is, at least in part, the consequence of the inhibition of K+ channels.

J Leukoc Biol, 2004 Oct, 76(4), 862 - 7 Epub 2004 Jul 26.
Insulin-like growth factor-I stimulates IL-10 production in human T cells; Kooijman R et al.; There is vast body of evidence that the insulin-like growth factor (IGF)-I exerts immunomodulatory effects in vitro and in vivo . In vitro studies indicate that stimulatory effects of IGF-I may be exerted through augmentation of inflammatory cytokine production . To further explore the immunomodulatory effects of IGF-I through regulation of cytokine production, we tested the in vitro effects of IGF-I on the secretion of inflammatory T helper cell type 1 (Th1) and Th2 cytokines by human peripheral blood mononuclear cells (PBMC) . To this end, PBMC were stimulated with the T cell mitogen phytohemagglutinin (PHA), and cytokines in the culture media were assessed after 18, 42, 66, and 80 h of culture . We found that IGF-I stimulated the secretion of the Th2 cytokine interleukin (IL)-10 by 40-70% in PHA-stimulated PBMC . In addition, we observed a small stimulatory effect (15%) on the secretion of another Th2 cytokine IL-4 . The secretion of IL-2, IL-5, IL-6, interferon-gamma, and the inflammatory cytokines IL-1beta, IL-8, and tumor necrosis factor alpha was not or was hardly affected . IL-10 secretion was also stimulated in purified T cells, and we established that IGF-I also stimulated IL-10 mRNA expression by 100-150% . The monocyte-activating bacterial cell-wall product lipopolysaccharide induced IL-10 production in PBMC, but this was not affected by IGF-I . As IL-10 predominantly exerts anti-inflammatory actions and suppresses Th1-dependent immune responses, our results indicate that IGF-I may exert inhibitory actions on inflammatory and Th1-mediated cellular immune responses through stimulation of IL-10 production in T cells.

Invest Ophthalmol Vis Sci, 2004 Aug, 45(8), 2586 - 95
In vitro antiangiogenic activity in ex vivo expanded human limbocorneal epithelial cells cultivated on human amniotic membrane; Ma DH et al.; PURPOSE: To compare the in vitro antiangiogenic activities of ex vivo expanded human limbocorneal epithelial (HLE) cells cultivated on preserved human amniotic membrane (AM) and to identify factors responsible for the activities . METHODS: The antiangiogenic effects were compared of culture media conditioned by AM, HLE cells, or HLE cells cultivated on intact AM (HLE/IAM), on denuded AM (HLE/DAM), or on DAM cocultured with 3T3 fibroblasts (HLE/DAM/3T3) . A monolayer culture of human umbilical vein endothelial cells (ECs) was used in a proliferation and migration assay . ECs suspended in type I collagen gel were used to assess capillary tube formation . Quantitative analyses of tissue inhibitor of metalloproteinase (TIMP)-1, thrombospondin (TSP)-1, pigment epithelium-derived factor (PEDF), and endostatin (proteolytic fragment of collagen XVIII) were performed by ELISA . Immunoconfocal microscopy was performed to localize the site of endostatin expression in HLE cells and AM . RESULTS: HLE cell- but not AM-conditioned medium (CM) inhibited the proliferation and migration of ECs, and coculture of HLE cells, but not of AM, with ECs inhibited capillary tube formation . Although some data from HLE cells alone are not significantly different from the control, increased inhibitory activity was expressed by HLE/IAM and HLE/DAM and was most significantly expressed by HLE/DAM/3T3 . Quantitation of TIMP-1, TSP-1, PEDF, and endostatin revealed that only the level of endostatin showed an increased expression by HLE cells cultivated on AM . Neutralizing antibody to endostatin substantially abrogated the inhibitory effect on EC proliferation and migration, but was less effective on EC differentiation . Endostatin signal was more prominent in the basement membrane zone of HLE cells cultivated on denuded AM than in those cultivated on intact AM . CONCLUSIONS: The antiangiogenic effect of HLE cells was enhanced when they were cultivated on AM and cocultured with 3T3 fibroblasts, and endostatin-related antiangiogenic factor may play a major role . This highlights the significance of cell-matrix and cell-cell interaction in the regulation of antiangiogenic factor secretion by HLE cells.

Diabetes, 2004 Aug, 53(8), 2018 - 23
Autocrine regulation of single pancreatic beta-cell survival; Navarro-Tableros V et al.; Function and survival of cells depend in part on the presence of growth factors . We explored the autocrine regulation of insulin and nerve growth factor (NGF) on single adult rat pancreatic beta-cell survival and hormone secretion . When NGF or insulin signaling were blocked in culture media, cell survival decreased compared with control cells, with apoptosis being the main mechanism of cell death . To further explore the role of glucose in beta-cell survival, we cultured the cells for 16 h in 2.6 mmol/l glucose and observed that nearly 17% of the cells developed apoptosis; this effect was partially prevented by NGF and almost completely inhibited by insulin treatment . A high K+ concentration had the same effect, suggesting that insulin and NGF secretion by the cells was responsible for the survival effects and not glucose per se . Blocking NGF signaling with an NGF antibody or with K252a reduced insulin biosynthesis and secretion in the cells that survived the treatment . Moreover, the functional beta-cell subpopulation with a higher insulin secretion rate is more susceptible to K252a . These results further indicate that NGF and insulin play important autoregulatory roles in pancreatic beta-cell survival and function and strongly suggest the need to explore new focuses in diabetes treatment.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 2911 - 7
Estimation of serum-free 50-percent inhibitory concentrations for human immunodeficiency virus protease inhibitors lopinavir and ritonavir; Hickman D et al.; Using measured free fraction and 50% inhibitory concentration (IC50) values for the human immunodeficiency virus protease inhibitors lopinavir (LPV) and ritonavir (RTV) in tissue culture media with various protein concentrations ranging from 5 to 50%, we estimated serum-free IC50 values for each drug . The range of serum-free IC50 values (0.64 to 0.77 ng/ml for LPV and 3.0 to 5.0 ng/ml for RTV) did not exhibit a trend with increasing protein concentrations, despite a 10-fold difference in the free fraction value (0.006 to 0.063) for LPV and a 5-fold difference in the free fraction value (0.013 to 0.057) for RTV . The mean serum-free IC50 by the MTT-MT4 assay (0.69 ng/ml for LPV and 4.0 ng/ml for RTV) may be the most accurate parameter for the estimation of the inhibitory quotient (IQ), a relative measure of in vivo potency defined as the ratio of the minimal free drug concentration in plasma (C(trough,free)) for a specific patient population and the serum-free IC50 . Using this approach, we calculated the average IQs for protease inhibitor-naive patients for LPV and RTV to be 67 and 5.6, respectively.

Microbiol Immunol, 2004, 48(7), 541 - 5
Autolysis of Porphyromonas gingivalis is accompanied by an increase in several periodontal pathogenic factors in the supernatant; Kamaguchi A et al.; Porphyromonas gingivalis autolyzes in the culture media . To examine in more detail the molecular components of the autolysate, two-dimensional gel electrophoresis was performed . Many protein spots varied both in number and volume . One of these spots included Arg-gingipain (Rgp) as determined by N-terminal amino acid sequencing . Corresponding to the increase in spot volume, Rgp activity also increased during autolysis . The results of this study suggested that Rgp and other proteins in the P . gingivalis autolysate may be involved with the prolongation of periodontal disease, even after the death of P . gingivalis cells.

Clin Exp Immunol, 2004 Aug, 137(2), 430 - 6
Osteoprotegerin (OPG) acts as an endogenous decoy receptor in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells; Miyashita T et al.; We examined the role of osteoprotegerin (OPG) on tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in rheumatoid fibroblast-like synovial cells (FLS) . OPG protein concentrations in synovial fluid from patients with rheumatoid arthritis (RA) correlated with those of interleukin (IL)-1beta or IL-6 . A similar correlation was present between IL-1beta and IL-6 concentrations . Rheumatoid FLS in vitro expressed both death domain-containing receptors {death receptor 4 (DR4) and DR5} and decoy receptors {decoy receptor 1 (DcR1) and DcR2} . DR4 expression on FLS was weak compared with the expression of DR5, DcR1 and DcR2 . Recombinant TRAIL (rTRAIL) rapidly induced apoptosis of FLS . DR5 as well as DR4 were functional with regard to TRAIL-mediated apoptosis induction in FLS; however, DR5 appeared be more efficient than DR4 . In addition to soluble DR5 (sDR5) and sDR4, OPG administration significantly inhibited TRAIL-induced apoptogenic activity . OPG was identified in the culture supernatants of FLS, and its concentration increased significantly by the addition of IL-1beta in a time-dependent manner . Neither IL-6 nor tumour necrosis factor (TNF)-alpha increased the production of OPG from FLS . TRAIL-induced apoptogenic activity towards FLS was reduced when rTRAIL was added without exchanging the culture media, and this was particularly noticeable in the IL-1beta-stimulated FLS culture; however, the sensitivity of FLS to TRAIL-induced apoptosis itself was not changed by IL-1beta . Interestingly, neutralization of endogenous OPG by adding anti-OPG monoclonal antibody (MoAb) to FLS culture restored TRAIL-mediated apoptosis . Our data demonstrate that OPG is an endogenous decoy receptor for TRAIL-induced apoptosis of FLS . In addition, IL-1beta seems to promote the growth of rheumatoid synovial tissues through stimulation of OPG production, which interferes with TRAIL death signals in a competitive manner.

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2004 Jul, 33(4), 290 - 5
{Protective effects of minocycline against hair follicle damage induced by cytosine arabinoside in vitro}; Wu XJ et al.; OBJECTIVE: To investigate the protective effects of minocycline against hair follicle damage induced by cytosine arabinoside (Ara-c) . METHODS: An in vitro organ culture of mouse vibrissa follicles was used and different concentrations of Ara-c and minocycline were added in the culture media . The total growth length, growth speed and growth period of hair were observed with invert microscopy and the survival of hair bulb cells was measured by MTT method . RESULT: Minocycline (0.3 x 10(-6) approximately 10(-5) mol/L) improved hair follicle total growth length, growth speed and hair growth period and also improved survival of hair bulb cells in vitro organ culture, which were inhibited by Ara-c . CONCLUSION: Minocycline can protect hair follicle directly from damage induced by Ara-c.

J Biomater Sci Polym Ed, 2004, 15(5), 645 - 60
Manufacture of elastic biodegradable PLCL scaffolds for mechano-active vascular tissue engineering; Jeong SI et al.; A soft and very elastic poly(lactide-co-epsilon-caprolactone) (PLCL)(50:50, Mn 185 x 10(3)) was synthesized . Tubular scaffolds were prepared by an extrusion-particulate leaching method for mechano-active vascular tissue engineering . The copolymer was very flexible but completely rubber-like elastic . Even the high porous PLCL scaffolds (90% salt wt) exhibited 200% elongation, but recovery over 85% in a tensile test . Moreover, the PLCL scaffolds maintained their high elasticity also in culture media under cyclic mechanical strain conditions . The highly porous scaffold (90% salt wt) withstood for an initial 1 week without any deformation and sustained for 2 weeks in culture media under cyclic stress of 10% amplitude and at 1 Hz frequency which are similar to the natural vascular conditions . Vascular smooth muscle cells (VSMCs) were seeded on to the PLCL scaffolds . The cell adhesion and proliferation on the scaffolds of various pore-size were increased with increasing pore size . For the pore sizes of 50-100 microm, 100-150 microm, 150-200 microm and 200-250 microm, the ratios of cell numbers were about 1:1.2:1.9:2.2, respectively, at both 12 h and 5 days . Similarly, the higher porous scaffolds exhibited more cell adhesion and proliferation compared to lower porous one, where the effect was more pronounced in the longer proliferation period . SMC-seeded scaffolds were implanted subcutaneously in athymic nude mice to confirm the biocompatibility . Such a high elastic property and proper biocompatibility to SMCs of PLCL scaffolds prepared in this study will be very useful to engineer SM-containing tissues such as blood vessels under mechanically dynamic environments (mechano-active tissue engineering).

Biomaterials, 2005 Jan, 26(3), 285 - 95
The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression; Chou YF et al.; The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5 x SBF) at 37 degrees C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures . Depending on processing parameters, different apatite structures can be created over the same substrate . The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression . MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS) . PreA induced significantly higher cell death during the first two weeks . TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA . Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation . By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR . The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.

Environ Res, 2004 Sep, 96(1), 62 - 71
In vitro effects on macrophages induced by noncytotoxic doses of silica particles possibly relevant to ambient exposure; Balduzzi M et al.; The RAW 246.7 macrophage cell line was exposed in vitro to aged crystalline silica particles of respirable size for 24 h at a range of doses starting from 15 microg/2 x 10(6) cells, which is a realistic exposure level of macrophages in the airways of ambiently exposed individuals . The particle sample used for the experiments was prepared to mimic some aspects of ambient crystalline silica particles: size distribution, morphology, and surface reactivity . Our purpose was to determine whether a nontoxic quartz load comparable to that of ambient exposure would be able to induce macrophage activation and impairment of the phagocytic ability, factors altering the lung's capacity to deal with increased particle loads (as occurs during high-pollution episodes) or infections and affecting the local and systemic responses through the release of biologically active compounds (cytokines, reactive oxygen species, NO, isoprostanes) . Exposure of RAW 264.7 cells to aged silica particles induced macrophage activation (evidenced by the morphological features observed with scanning electron microscopy and by the release of TNF-alpha and IL-6) and impairment of phagocytosis of test particles, even at noncytotoxic doses . The reduction of the phagocytic function of the cells after silica treatment was dose-dependent, as evidenced by an increase of the population of unphagocytic cells, paralleled by a decrease of the actively phagocytizing cell population . We evaluated the oxidative stress induced by aged silica particles, quantifying the peroxidation products (8-isoprostanes) in the culture media of treated cells, and found a strong release at low doses . Isoprostanes are a complex family of compounds which have been used as in vivo markers of lipid peroxidation in human disorders, but that, as far as we know, have never been evaluated in relation to airborne particulate matter exposure . Lipid peroxides are involved in various cellular events in the inflammatory response, and isoprostanes are also supposed to exert important biological actions on airway and pulmonary vascular smooth muscles and on platelets.

Antivir Ther, 2004 Jun, 9(3), 353 - 63
In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir; Lada O et al.; Emergence of lamivudine-resistant hepatitis B virus (HBV) is a major concern in human immunodeficiency virus (HIV) and HBV coinfected patients . Following selection of resistant mutants, hepatitis flare or rapid progression to cirrhosis may occur . Treatment of patients with new nucleotide analogues such as adefovir dipivoxil (ADV) or tenofovir disoproxil fumarate (TDF) has shown good efficacy in controlling wild-type or lamivudine-resistant HBV replication . The purpose of this study was to assess the in vitro efficacy of new nucleotide analogues on HBV strains isolated from lamivudine-treated patients . After purification of HBV DNA from patient sera, the whole HBV genome was PCR-amplified and cloned . Drug sensitivity was measured after transfection of the isolated full genomes into HepG2 cells and measurement of HBeAg, HBsAg and viral replication in the culture media under increasing drug concentrations . A wild-type strain isolated from an untreated patient served as control . In a clinical study of ADV (Gilead 460i study), seven of the 35 patients carried HBV strains with the triple lamivudine resistance-associated amino-acid changes rtV173L/L180M/M204V at baseline . Although all patients responded to ADV in this clinical study, the serum HBV reduction was lower in the seven patients with the triple mutation (median -3.3 log copies/ml) compared to the patients who had only the rtL180M/M204V mutations (median -4.1 log copies/ml) at week 48 (P=0.04, Mann-Whitney test) . In our in vitro system, lamivudine IC50 on lamivudine-resistant HBV carrying amino-acid substitutions rtL180M and rtM204V within the polymerase encoding region increased by more than 16,000-fold (from 6 nM to over 100 microM) when compared to wild-type HBV . For ADV and TDF, comparison of wild-type and lamivudine-resistant HBV IC50 (rtL180M-M204V) showed, respectively, 2.85-fold (from 0.07 to 0.2 microM) and 3.3-fold (from 0.06 to 0.2 microM) increases, indicating a mild decrease of both drug activities, in vitro . At the ADV concentration of 0.1 microM, presence of the V173L mutation reduced the inhibition of HBsAg production from 50 to 30% (P<0.01) and the viral replication from 45 to 32% (P<0.01, Mann-Whitney) . Conversely, tenofovir had similar potency on both HBV mutation profiles with 60% inhibition of HBsAg production and 45% inhibition of viral replication at 0.1 microM . Our study supports the high efficacy of ADV and TDF seen in patients after lamivudine breakthrough . The excellent activity of TDF on lamivudine-resistant virus independently of the resistance mutation profile offers an interesting treatment alternative to HIV-HBV coinfected patients.

Reprod Biomed Online, 2004 Jul, 9(1), 70 - 3
Discrepancies between the effects of glutamine in cultures of preimplantation mouse embryos; Biggers JD et al.; A review of the literature shows divergent differences between laboratories of the effects of glutamine in mouse preimplantation embryo culture media . One laboratory reported several cases of exencephaly, which was attributed to ammonia produced by the breakdown of glutamine . Two other laboratories have found no such effects . It is suggested, but not proved, that the differences in results may have a genetic basis . Further, it is argued that studies on the toxicological actions of exogenous ammonium chloride on preimplantation development provide a biased model of the effects of glutamine as used in embryo culture protocols . The finding that ammonium can also cause exencephaly thus fosters undue concern about the teratological effects of glutamine.

Appl Microbiol Biotechnol, 2004 Sep, 65(4), 479 - 87 Epub 2004 Jul 15.
Degradation of acrylic copolymers by white-rot fungi; Mai C et al.; Various water-soluble homopolymers and copolymers of acrylamide (AAm) and acrylic acid (AA) which contained phenolic sites, such as guaiacol, lignin sulfonate (LS) and 3,4-dihydroxybenzoic acid (3,4-DHBA), were tested with regard to their degradability by white-rot fungi . Compared with Phanerochaete chrysosporium, Pleurotus ostreatus caused a significantly higher decrease in the average molecular weight ( Mw) of most of the copolymers and the homopolymer under the applied culture conditions . However, the Mw of poly(guaiacol/AAm) increased significantly during incubation with Pl ostreatus . P . chrysosporium was able to reduce only the Mw of the poly(LS/AA) to a significant degree and not that of the other polymers . The mineralization rate of AAm and AA copolymers and terpolymers of AAm, AA and phenolics (LS, 3,4-DHBA, guiacol), which were tested with P . ostreatus and Trametes versicolor, turned out to be low (0.8-3.2%) . While the rates of mineralization were similar among all polymers, the decrease in radioactivity from the culture media was higher with the terpolymers bearing phenolic sites . UV spectra of the culture media revealed that the phenolic sites in the terpolymers were significantly degraded by both fungi . Obviously, the degradation of phenolics within the polymer chain caused a higher decrease in Mw but did not significantly increase the mineralization rate.

Pancreas, 2004 Aug, 29(2), 104 - 9
Caspase-3 inhibitor prevents apoptosis of human islets immediately after isolation and improves islet graft function; Nakano M et al.; OBJECTIVES: Apoptosis appears in islets after isolation, and it has a detrimental effect on the islet function . To improve the outcome of clinical islet transplantation, it is crucial to protect islets from apoptosis . The aim of this study was to determine whether a caspase-3 inhibitor (Z-DEVD-FMK) added to culture media protects islets from apoptosis and to compare the effects of fetal bovine serum (FBS) with human serum albumin (HSA) as a protein supplement in culture . METHODS: Isolated human islets were cultured under 4 different conditions: 0.5% HSA (control), 0.5% HSA + 25 micromol/L Z-DEVD-FMK, 0.5% HSA + 100 micromol/L Z-DEVD-FMK and 10% FBS for 2 days . Next, 1000 IEQ islets precultured with 0.5% HSA and with or without 100 micromol/L Z-DEVD-FMK were transplanted to diabetic nude mice . RESULTS: The islet yields were higher in Z-DEVD-FMK-treated groups, and the inhibitor prevented apoptosis dose dependently . The yield and insulin release were higher in FBS-treated group than in the control group, but FBS did not affect apoptosis . All 6 mice transplanted with islets pretreated with Z-DEVD-FMK, and 3 of 8 mice with control islets became normoglycemic posttransplantation . CONCLUSION: Z-DEVD-FMK prevented apoptosis of isolated human islets and improved its function . FBS (10%) improved the islet yield and insulin secretion more than 0.5% HSA.

Cell Tissue Bank, 2001, 2(1), 15 - 21
University of wisconsin solution with trypsin inhibitor pefabloc improves survival of viable human and primate impure islets during storage; Matsumoto S et al.; Background . Recent studies suggest that impure islets (islets which have not been isolated from exocrine tissue and other parts of the pancreas) have great potential for successful transplantation . The evidence that supports this view includes findings that embedded islets (islets surrounded by exocrine tissue) undergo less apoptosis, peripancreatic lymph nodes prevent recurrence of IDDM (insulin dependent diabetes mellitus), and that islet yields and insulin content decrease during the purification process . Improved protocols have also been developed to prevent allorejection of impure islets . Despite these promising results, the storage of impure islets remains difficult, and was a method sought to decrease storage losses.Methods . Storage methods of impure human and non-human primate islets were compared, using either culture media or University of Wisconsin solution (UW) . The effects of trypsin inhibition using Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN) during storage period were also examined.Results . Low temperature and inhibition of trypsin activity during storage of impure islets improved both islet yield and viability . It was found that using UW solution and trypsin inhibition allowed perfect preservation of viable impure islets up to 48 h . A functional assay by glucose stimulation test showed these impure islet responded to glucose stimulation after 24 h.Conclusion . The benefits of storing impure islets using UW solution and Pefabloc at low temperature have been established . This improved method of preserving impure islets makes this model of transplantation even more viable.

Biochem J, 2004 Nov 1, 383(Pt . 3), 507 - 15
Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation; Gopalakrishnan MM et al.; Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes . We demonstrate that such complexes are also present in secretions of cultured cells . To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs . The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids . In gel filtration, proSAP behaved as an oligomer and proCD as a monomer . ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions . ProSAP did not change the elution of mature cathepsin D . Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions . At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer . The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer) . ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D . Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments . Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D . In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.

FEBS Lett, 2004 Jul 16, 570(1-3), 73 - 6
Secretion of long Abeta-related peptides processed at epsilon-cleavage site is dependent on the alpha-secretase pre-cutting; Kametani F; Abeta is the major component of amyloid in the brain in Alzheimer's disease and is derived from Alzheimer amyloid precursor protein (APP) by sequential proteolytic cleavage involving alpha-, beta- and gamma-secretase . Recently, gamma-secretase was shown to cleave near the cytoplasmic membrane boundary of APP (called the epsilon-cleavage), as well as in the middle of the membrane domain (gamma-cleavage) . However, the precise relationship between gamma- and epsilon-cleavage is still unknown . In this paper, I analyzed Abeta-related peptides using immunoprecipitation and liquid chromatography ion trap mass spectrometer and found some long Abeta-related peptides, starting at Abeta residues 16Lys-23Asp and ending at 43Thr-52Leu, in the culture media of COS-1 cells and in human brain extract . These results indicated that longer Abeta-related peptides cleaved at epsilon-cleavage site were secreted under normal conditions and were dependent on the alpha-secretase cleavage products.

Toxicol In Vitro, 2004 Oct, 18(5), 691 - 701
A short-term in vitro gill culture system to study the effects of toxic (copper) and non-toxic (cortisol) stressors on the rainbow trout, Oncorhynchus mykiss (Walbaum); Mazon AF et al.; A short-term (24 h) method of gill filament culture system was developed to predict the effects of environmental contamination and stress in fish . Gill culture system containing two or three rainbow trout gill filaments in sterile glutamine supplemented Leibovitz 15 (L-15) media was submitted for 24 h to six different treatments: (i) CONT (control, medium only); (ii) CORT (cortisol, 0.28 microM cortisol); (iii) BLOCK (glucocorticoid receptor blocker, 14 microM RU 486); (iv) CORT+BLOCK (cortisol and blocker, 0.28 microM cortisol+14 microM RU 486); (v) CORT+CU (cortisol and copper, 100 microM CuSO4+0.28 microM cortisol); (vi) CU (copper, 100 microM CuSO4) . After 24 h, the overall gill structure and cellular components resembled those of salmonids in vivo . Lactate dehydrogenase (LDH) activity in the culture media increased in the CORT+CU and CU groups but was significantly lower in the CORT+CU compared to CU group . Apoptotic cells increased in the CORT and CORT+BLOCK . The numbers of glucocorticoid (GR) receptor-positive cells were lower in the CU group . This short-term culture system seems to be suitable for studying the effects of both external and internal stress effectors (toxicants and hormones respectively), as it contains all cell types found in the gills and the cells give similar biological response as in vivo.

Toxicol In Vitro, 2004 Oct, 18(5), 593 - 9
An inhibitor of p38 MAP kinase downregulates cytokine release induced by sulfur mustard exposure in human epidermal keratinocytes; Dillman JF 3rd et al.; Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a potent alkylating agent that induces skin vessication after cutaneous exposure . Previous work has revealed that SM induces the production of inflammatory cytokines, including IL-8, IL-6, TNF-alpha, and IL-1beta, in keratinocytes . The p38 MAP kinase (MAPK14) signaling pathway is activated via phosphorylation in response to cellular stress and has been implicated in the upregulation of cytokines in response to stress . We investigated the role of p38 MAP kinase in inflammatory cytokine upregulation following SM exposure . A dose response study in cultured human epidermal keratinocytes (HEK) revealed increasing phosphorylation of p38 MAP kinase in response to increasing concentrations of SM . A time course at the 200 microM exposure revealed that p38 MAP kinase phosphorylation is induced by 15 min post-exposure, peaks at 30 min and is sustained at peak levels until 8 h post-exposure . Phosphorylation of the upstream kinase MKK3/6 was also detected . Assay of the SM-exposed HEK culture media for cytokines revealed that exposure to 200 microM SM increased IL-8, IL-6, TNF-alpha, and IL-1beta . When cells exposed to 200 microM SM were treated with the p38 MAP kinase inhibitor SB203580, the levels of IL-8, IL-6, and TNF-alpha and IL-1beta were significantly decreased when compared with cells that were untreated . These results show that p38 MAP kinase plays a role in SM-induced cytokine production in HEK and suggest that inhibiting this pathway may alleviate the profound inflammatory response elicited by cutaneous SM exposure.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Jun, 21(3), 363 - 6
{Force-dependent effects of interleukin-8 production in endothelial cells exposed to fluid shear stress}; Tang R et al.; Fluid shear stress plays a key role in many physiological activities and pathological processes of the cardiovascular diseases . In vivo, endothelial cells (ECs) are constantly exposed to hemodynamic force which can modulate structure and function of ECs . Previous studies have demonstrated that IL-8 protein production in endothelial cells was modulated by fluid shear stress, and IL-8 protein production induced by fluid shear stress was time-dependent . In order to identify the role of intensity of fluid shear stress on IL-8 protein production of human umbilical vein endothelial cells (HUVECs), we had HUVECs exposed to shear stress 2.09, 4.61, 6.19, 8.51, 10.50, 12.59, 14.41, 17.22, 18.32 dyne/cm2 respectively and employed quantitative sandwich enzyme-linked immunosorbent assay (ELISA) to measure the IL-8 protein . Here we show that HUVECs untreated with fluid shear stress secreted very little IL-8 in culture media . The IL-8 protein production induced by shear stress was force intensity-dependent . After HUVECs being subjected to low fluid shear stress (2.09 dyne/cm2) for 5 h or 6 h, IL-8 protein production increased and was nearly 6 times or 7 times over that of HUVECs subjected to high fluid shear stress (18.32 dyne/cm2) . The linear regression equations between IL-8 protein production (y) and shear stress (dyne/cm2, x) are y=760.12-36.06x, gamma=-0.978 (for 5 h); y=781.87-36.66x, gamma=-0.980 (for 6 h) . This in vitro study demonstrates that the production of IL-8 can be regulated by fluid shear stress, and the production of IL-8 induced by shear stress is not only time-dependent but also force intensity-dependent . These observations suggest that the low fluid shear stress induces much more IL-8 secretion, which may play an important role in the pathogenesis and development of both inflammation and atherosclerosis.

J Neurosci Res, 2004 Aug 1, 77(3), 354 - 62
Enhanced production and proteolytic degradation of insulin-like growth factor binding protein-2 in proliferating rat astrocytes; Chesik D et al.; Insulin-like growth factors (IGFs) protect neurons, are important for oligodendrocyte survival and myelin production, and stimulate the proliferation of astrocytes . The effects of IGFs are regulated by a family of IGF binding proteins (IGFBPs) . Astrocytes express predominantly IGFBP-2 . In the present study, primary neonatal rat astrocytes were cultivated in a chemically defined medium to initiate a differentiated cell status . After stimulation with fetal calf serum, astrocytes became hypertrophic and increased proliferation . Western blot analysis of cell lysate of proliferating astrocytes displayed an increased expression of IGFBP-2 . This finding was supported by immunocytochemical images . Semiquantitative polymerase chain reaction analysis demonstrated equal mRNA levels in both differentiated and proliferating astrocytes, suggesting that the increase in IGFBP-2 production in proliferating astrocytes was exerted at the translational level . Concentrated medium of proliferating cells, however, displayed lower levels of IGFBP-2 than differentiated cells . When recombinant IGFBP-2 was incubated with culture media, we found degradation in the medium of proliferating cells, but not in medium of differentiated cells . This degradation could be inhibited with protease inhibitors, indicating that lower levels of IGFBP-2 in the medium of proliferating astrocytes are due to the presence of proteases . Our results suggest that, in proliferating astrocytes, IGFBP-2 may help target IGFs to IGF-1 receptors, and IGFBP-2 proteases may play a role in enhancing the availability of IGFs .

Exp Neurol, 2004 Aug, 188(2), 408 - 20
Heparin stabilizes FGF-2 and modulates striatal precursor cell behavior in response to EGF; Caldwell MA et al.; Fibroblast and epidermal growth factors (FGF-2 and EGF) are powerful mitogens for neural precursor cells isolated from the developing striatum and grown as neurospheres . However, questions remain as to the exact role of each of these molecules, and how the proteoglycan heparin may modify their behavior . Here, we show that FGF-2 is remarkably unstable in culture media, but that heparin could completely prevent its degradation, which led to faster cell growth rates . In addition, heparin significantly increased the number of cells within the E14 striatum responding to a brief pulse of FGF-2 . In contrast, EGF was unable to stimulate the growth of E14 striatal precursors . However, EGF could induce the division of E18 striatal precursors as neurospheres and acted synergistically with FGF-2 . FGF-2/heparin neurospheres generated significantly more neurons than EGF neurospheres . Interestingly, the addition of heparin to EGF neurospheres, which had no effects on EGF stability or growth rates, increased the numbers of neurons generated to that seen for FGF-2/heparin neurospheres . EGF neurospheres were found to produce FGF-2, but addition of heparin did not affect its concentration within cells or in the medium suggesting this released FGF-2 may already be bound to a proteoglycan . In addition, expanding cells with EGF plus heparin in the presence of an FGF-2 blocker did not have a significant effect on the number of neurons generated confirming that the increase in neuronal number is through a mechanism which is independent of FGF-2.

Lancet, 2004 Jul 10, 364(9429), 163 - 71
Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro; Zhan X et al.; BACKGROUND: Differentiated cells derived from pluripotent human embryonic stem (hES) cells offer the opportunity for new transplantation therapies . However, hES cells and their differentiated progeny express highly polymorphic MHC molecules that serve as major graft rejection antigens to the immune system of allogeneic hosts . To achieve sustained engraftment of donor cells, strategies must be developed to overcome graft rejection without broadly suppressing host immunity . One approach entails induction of donor-specific immune tolerance by establishing chimeric engraftment in hosts with haemopoietic cells derived from an existing hES cell line . We aimed to develop methods to efficiently differentiate hES cells to haemopoietic cells, including immune-modulating leucocytes, a prerequisite of the tolerance induction strategies applying to hES cell-mediated transplantation . METHODS: We developed a method to generate a broad range of haemopoietic cells from hES-generated embryonic bodies in the absence of murine stromal feeder cells . Embryonic bodies were further cultured in the presence of haemopoietic cytokines . In addition to flow cytometric analyses of haemopoietic cell markers, we analysed the hES cell-derived haemopoietic cells by colony-forming assays (for erythroid and myeloid progenitor cells), cytochemical staining, and mixed leucocyte reactions to determine the functional capacity of the generated antigen-presenting cells . FINDINGS: 12 independent experiments were done . When selected growth factors were added, leucocytes expressing CD45 were generated and released into culture media for 6-7 weeks . Under the condition used, both erythroid and myeloid progenitor cells were generated . About 25% of the generated leucocytes acquired MHC class II and costimulatory molecule expression . These hES-derived, MHC class II+ leucocytes resembled dendritic cells and macrophages, and they functioned as antigen-presenting cells capable of eliciting allogeneic CD4 and CD8 T-cell responses in culture . INTERPRETATION: The hES cell-derived antigen-presenting cells could be used to regulate alloreactive T cells and induce immune tolerance for improvement of the transplant acceptance of hES-cell derivatives.

Diabetologia, 2004 Jul, 47(7), 1167 - 74 Epub 2004 Jul 09.
Mutation at position -132 in the islet amyloid polypeptide ( IAPP) gene promoter enhances basal transcriptional activity through a new CRE-like binding site; Novials A et al.; AIMS/HYPOTHESIS: Mutations in the islet amyloid polypeptide ( IAPP) gene may play a potential role in the abnormal regulation or expression of the peptide . The aim of this study was to determine the functional role of the -132 G/A mutation reported in the promoter region of the IAPP gene in a population of Spanish Type 2 diabetic patients . METHODS: We investigated the transcriptional activity using MIN6 cells and luciferase reporter plasmids in several culture conditions . Key regulatory elements of the IAPP promoter region were also analysed by electrophoretic mobility shift assays (EMSA) . RESULTS: The mutant construct doubled IAPP transcriptional activity ( p<0.001) . Both constructs showed severely reduced promoter activity (four-fold decrease) in the presence of verapamil and diazoxide . In contrast, IAPP promoter activity was doubled after incubation with forskolin or dexamethasone, regardless of the glucose concentrations in the culture media . EMSA revealed that the -132 G/A mutation increased the binding affinity through two DNA-protein complexes . In addition, a cAMP-responsive element binding protein (CREB) was identified by super-shift EMSA . CONCLUSIONS/INTERPRETATION: Our studies show that the wild-type and the mutant constructs are regulated in a similar pattern under all conditions, strongly indicating that the -132 G/A mutation increases basal but not inducible transcription . These results may be explained by new binding to the mutant region through CREB and other transcription factors not yet identified.

Hum Reprod, 2004 Sep, 19(9), 2109 - 17 Epub 2004 Jul 08.
Chromosomal abnormality rate in human pre-embryos derived from in vitro fertilization cycles cultured in the presence of Follicular-Fluid Meiosis Activating Sterol (FF-MAS); Bergh C et al.; BACKGROUND: The objective of the study was to investigate the effect of Follicular-Fluid Meiosis Activating Sterol (FF-MAS) when added to the culture media on the incidence of chromosomal abnormalities and pre-embryo development in human pre-embryos . METHODS: 243 women undergoing IVF/ICSI treatment donated 353 oocytes in a multicentre, prospective, randomized, double blind, four-arm, controlled trial performed at Danish and Swedish public and private IVF centers . Metaphase II oocytes were randomly assigned to: FF-MAS 5 microM, FF-MAS 20 microM, ethanol 0.2% (vehicle control) or water for injection (inert control) . The exposure regimen of FF-MAS to the human oocytes was 4 h prior to fertilization by ICSI and 20 h exposure post ICSI . The primary endpoint was the incidence of numerical chromosomal abnormalities . Secondary endpoints were cleavage rate and pre-embryo quality . RESULT: On the pre-embryo level, no significant differences in chromosomal abnormality rate were observed among the four groups . However, the percentage of uniformly normal pre-embryos was significantly lower in the pooled FF-MAS group (5 microM: 12% and 20 microM: 17%) than in the pooled control group (inert control 32% and vehicle control 42%) . A high level of mosaicism (41-60%) was found in all groups . At the blastomere level, the percentage of blastomeres categorized as normal was significantly lower in the FF-MAS 5 microM group (41%) and the FF-MAS 20 microM (29%) group versus the inert (52%) and the vehicle (61%) groups . Significantly reduced cleavage and good quality pre-embryo rates were found in both FF-MAS groups . CONCLUSION: FF-MAS increased the rate of aneuploidy and had detrimental effects on cleavage and pre-embryo development, when exposed both before and after fertilization.

World J Gastroenterol, 2004 Jul 15, 10(14), 2050 - 4
Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells; Ni Q et al.; AIM: To investigate the role of human La protein in HBV mRNA expression . METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR . After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting . Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line . HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR . RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively . In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression . Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control . Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs . CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein . The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.

Neuroreport, 2004 Jul 19, 15(10), 1633 - 7
Angiogenesis-related factors derived from retinal glial (Müller) cells in hypoxia; Eichler W et al.; Retinal glial (Muller) cells may play a major role in vascular eye diseases as they secrete vascular endothelial growth factor (VEGF), a hypoxia-induced angiogenic cytokine . They also release significant amounts of the anti-angiogenic factors, transforming growth factor (TGF)-beta2, pigment epithelium derived factor (PEDF), and thrombospondin-1 (TSP-1) . Exposure of human (MIO-M1) and guinea-pig Muller cells to hypoxia resulted in a decreased release of TGF-beta2 and PEDF but in an elevated secretion of TSP-1 . When retinal endothelial cells were exposed to VEGF/anti-angiogenic factor ratios mimicking those found in culture media of Muller cells under normoxia or hypoxia, their proliferation was significantly inhibited by TGF-beta2, PEDF or TSP-1 . Thus Muller cells may provide a permanent anti-proliferative condition for retinal endothelial cells.

Mycol Res, 2004 May, 108(Pt 5), 489 - 97
Culture studies and secondary compounds of six Ramalina species; Cordeiro LM et al.; Mycobiont isolation experiments were performed on six species of Ramalina from Brazil: R . celastri, R . complanata, R . dendriscoides, R . gracilis, R . peruviana and R . sprengelii . This study aimed to optimize the culture conditions and nutrient requirements of the selected mycobionts . The aposymbiotic R . complanta was successfully isolated from ascospores, while aposymbiotic R . peruviana was obtained from thallus fragments . In R . peruviana the production of secondary metabolites was investigated under aposymbiotical growth conditions using HPLC . When cultivated on solid medium, this mycobiont produced the typical chemosyndrome (sekikaic acid and satellite compounds), found in the voucher lichen thallus . When cultivated in liquid medium (immersed in malt yeast medium in the absence of agar), only one, the major lichen substance, sekikaic acid, was synthesized by the fungus . In addition, atranorin was formed, but was not detected in any of the voucher specimens . Red pigments were found in solid and liquid cultures . These were separated into two compounds, but could not be fully identified . R . celastri spores germinated, but did not form mycelia . R . dendriscoides, R . gracilis and R . sprengelii were not successfully cultivated in aposymbiotic conditions, although eight different culture media were tested.

J Nutr Sci Vitaminol (Tokyo), 2004 Feb, 50(1), 38 - 44
Inhibitory effect of coffee on hepatoma proliferation and invasion in culture and on tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats; Miura Y et al.; We have already reported that instant coffee powder (ICP) and ICP-loaded rat sera could suppress proliferation and invasion of rat ascites hepatoma cell line of AH109A in vitro . In this report, we examined the mechanisms for suppression of tumor cell proliferation and invasion by ICP, and the effect of ICP on in vivo tumor growth, metastasis and abnormal lipoprotein profiles in hepatoma-bearing rats . ICP, when directly added to the culture media, induced cell cycle arrest (elongation of S phase) at a lower concentration (0.3 mg/mL) and apoptosis at a higher concentration (0.6-1.2 mg/mL) . ICP and ICP-loaded rat sera showed reactive oxygen species (ROS)-scavenging property and canceled the enhancement of invasive activity of hepatoma cells induced by ROS in vitro . These results suggest that ICP suppresses the proliferation by inducing cell cycle arrest and apoptosis, and the invasion by scavenging ROS and that ICP could retain these properties after their gastrointestinal absorption . The hepatoma-bearing rats were fed with a 20% casein diet (20C) or 20C supplemented with 0.1%, ICP for 14 d . Dietary ICP significantly reduced solid tumor growth and tended to reduce hepatoma metastases to lung and lymphatic nodes, suggesting that ICP could suppress tumor cell proliferation and invasion in vivo . In addition, dietary ICP significantly increased serum high-density lipoprotein (HDL)-cholesterol and tended to reduce very low-density and low-density lipoprotein (VLDL+LDL)-cholesterol, resulting in amelioration of abnormal lipoprotein profiles occurred in hepatoma-bearing rats . In conclusion, ICP has the ability to induce cell cycle arrest and apoptosis in hepatoma cells and to suppress tumor cell invasion by reducing oxidative stresses in vitro, and it could also exhibit these effects in vivo, leading to the inhibition of tumor growth and metastases.

J Nutr, 2004 Jul, 134(7), 1716 - 23
Investigation of lymphocyte gene expression for use as biomarkers for zinc status in humans; Andree KB et al.; A bioassay for zinc status in humans has been sought due to the importance of zinc, an essential trace metal, for many divergent functions in the human body; however, a sensitive bioassay for zinc status in humans is lacking . To address this issue, we established gene expression profiles of human lymphoblastoid cells treated with 0 or 30 micro mol/L ZnSO(4) using microarray technology . A limited number of genes were responsive to 30 micro mol/L zinc based on the analysis of Affymetrix human genome U133A GeneChips . We also examined the gene expression patterns of zinc transporters in human lymphoblastoid cells using quantitative RT-PCR analysis . ZNT1 was upregulated in lymphoblastoid cells, whereas ZIP1 was downregulated in response to the increased zinc concentrations in the culture media . To evaluate the potential applications of using both zinc transporter genes as biomarkers of zinc status, we measured the expression levels of ZIP1 and ZNT1 in the peripheral leukocytes collected from 2 different age groups of Korean women . After administration of a zinc supplement (22 mg zinc gluconate/d for 27 d), ZIP1 expression decreased by 17% (P < 0.01) and 21% (P < 0.05) in the peripheral leukocytes collected from 15 young (20-25 y) and 10 elderly (64-75 y) subjects, respectively . ZNT1 expression was not affected by taking the zinc supplement . These data suggest a potential application of ZIP1 as a biomarker of zinc status in humans.

Sheng Li Xue Bao, 2004 Jun 25, 56(3), 288 - 94
{Effects of histamine on endothelial nitric oxide synthase expression in pulmonary artery endothelial cells}; Lu DQ et al.; All three nitric oxide synthase (NOS) isoforms are found in the lungs . It has been demonstrated that eNOS-derived NO plays an important role in modulating pulmonary vascular tone and inhibiting pulmonary vascular remodeling . Histamine induces pulmonary vasoconstriction by activating H(1)-receptor on the smooth muscle cells and vasodilation by stimulating H(2)-receptor . It remains unclear whether histamine also modulates the pulmonary vascular tone by regulating eNOS gene expression and NO production in pulmonary artery endothelial cells . Therefore, the present study was performed on cultured primary porcine pulmonary artery endothelial cells (PAECs) to investigate the effects of histamine on eNOS gene expression, and to explore the role of CaMK II in eNOS gene expression . After treatment with different concentrations histamine for different times, the levels of eNOS mRNA and protein were measured by RT-PCR and Western blot, respectively . The results showed that histamine upregulated eNOS mRNA and protein levels in a concentration- and time-dependent manner . Incubation with 10 micromol/L histamine for 24 h could increase eNOS mRNA and protein level to 160.8+/-12.2% (P<0.05) and 136.2+/-11.2% (P<0.05), respectively, of the control values . These up-regulation effects were prevented by selective CaMK II inhibitor, KN-93 (10 micromol/L) . To investigate whether or not histamine increases eNOS expression by upregulating eNOS gene transcription, PAECs were transiently transfected with 1.6-kb fragment of the human eNOS promoter driving a luciferase reporter gene . The results suggested that eNOS gene promoter activity was enhanced to 148.2+/-33.7% (P<0.05) of the control after PAECs were incubated with 10 micromol/L histamine for 24 h . The nitrite and nitrate content in culture media measured by colorimetric method after incubation with 10 micromol/L histamine for 24 h indicated that the NO production in PAECs was increased . These results suggest that histamine up-regulates eNOS gene transcription and enhances NO production in PAECs by a signaling pathway involving CaMK II, which might be one of the mechanisms of histamine modulating pulmonary vascular tone.

Ginecol Obstet Mex, 2003 Nov, 71, 551 - 8
{Properties of the chorioamnios zone inducing premature membranes rupture}; Meraz Cruz MC et al.; Premature membrane rupture (PMR) is one of the most serious public health problems in the world, ocurring in 10% of all pregnancies . PMR has important adverse effects on maternofetal morbidity-mortality, as it has been estimated that it accounts on the whole for 70% and 40% of neonatal morbidity and mortality, respectively . PMR treatment is empirical, as its aetiology is unknown and its physiopathogenic description has just been initiated . This work analyzes the possibility of documenting functional differences in human chorio-amnios, comparing the zone where rupture most frequently occurs in PMR with some other distant chorio-amnionic zones and with equivalent zones of fetal membranes obtained from nine month pregnancies which have not undergone labor . The membrane zone which was nearest to the cervical os was identified and marked to be analyzed later for extracellular matrix metalloprotease (MMP) activity, histology and topographical MMP distribution . The MMP expression was quantitatively determined in explant culture media from membrane fragments using specific immuno-enzymatic essays (ELISA) and zymography . In addition, immuno-histochemistry methods were used to reveal MMP expression in the different tissues . This methods allowed us to show the existence of a decreasing MMP activity gradient, with the greatest value corresponding to the zone nearest to the cervical os in the membranes obtained from PMR cases . In membranes obtained from cesarean operations no characteristic pattern was documented and values were always lower than those obtained for PMR tissues . We conclude that there is a chorio-amnionic zone in which connective tissue degradation is specifically induced and which coincides with the membrane zone in contact with the cervical os.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 46 - 55 Epub 2004 May 20.
Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity; Suarez B et al.; Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism . Several proteins with proteolytic activity were detected in culture filtrates from T . harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity . The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7-4.9 . The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity . Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1 . The protein sequence showed <43% identity to mammal trypsins and 47-57% to other fungal trypsin-like proteins described thus far . Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media . The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations . This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T . harzianum CECT 2413 . A role for PRA1 in the protection of plants against pests and pathogens provided by T . harzianum CECT 2413 is proposed.

Osteoarthritis Cartilage, 2004 Jul, 12(7), 577 - 85
The spread of cell death from impact damaged cartilage: lack of evidence for the role of nitric oxide and caspases; Clements KM et al.; Over 21 days in culture, cell death spreads, both radially and transversely, from loaded to surrounding cartilage . This spread was prevented by physical separation and separate culture post-impact . OBJECTIVE: One aim was to determine if nitric oxide (NO) is the intercellular signal mediating cell death . Another aim was to clarify the nature of the cell death, whether caspase mediated apoptosis or necrosis . DESIGN: Cyclic impacts were applied to the central 2 mm core of 4 mm canine articular cartilage discs . Post-impact culturing was for 21 days in the presence or absence of the iNOS inhibitor, L-NAME, or the broad-spectrum caspase inhibitor, Z-VAD FMK . Cell death was quantified using the TUNEL assay . Culture media were collected every 2 days for measurements of glycosaminoglycan (GAG) and NO release . RESULTS: Cell death spread from the loaded core into the surrounding ring over 21 days in culture . Although L-NAME significantly reduced nitrite release into the culture media of both loaded and control cartilage, the spread of cell death was not prevented . Neither was the spread of cell death prevented by Z-VAD FMK . CONCLUSIONS: These data indicate that NO is not acting as an intercellular signalling factor in this in vitro system and that the cell death post-impact is not caspase mediated .

Eur Cytokine Netw, 2004 Jan-Mar, 15(1), 6 - 13
Interleukin-3 and ex vivo maintenance of hematopoietic stem cells: facts and controversies; Ivanovic Z; Although the utilization of IL-3 in the ex vivo expansion of hematopoietic stem cells has been considered as an attractive possibility, its mode of action remains unclear and controversial . Some reports show that IL-3 maintains or even enhances primitive stem cell activity, whereas others show the opposite . The presence of serum in culture media enhances the pro-differentiating effect of IL-3 on stem cells . Conversely, addition of IL-3 to serum-free cultures improves the capacity of TPO, SCF and Flt3-ligand to promote the self-renewal of primitive stem cells . The presence or absence of serum or of some serum substitutes (in serum-free cultures), as well as other culture parameters are probably responsible for these contrasting effects of IL-3 on stem cells . However, none of the data presently evaluated bring a clear, definitive explanation to this apparent paradox . Those data that appear to be the most informative are presented and discussed in this "technical review".

J Comp Neurol, 2004 Jul 19, 475(2), 211 - 9
Human neural stem cell transplants improve motor function in a rat model of Huntington's disease; McBride JL et al.; The present study investigated the neuroanatomical and behavioral effects of human stem cell transplants into the striatum of quinolinic acid (QA)-lesioned rats . Twenty-four rats received unilateral QA (200 nM/microl) injections into the striatum . One week later, rats were transplanted with stem cells derived from human fetal cortex (12 weeks postconception) that were either 1) pretreated in culture media with the differentiating cytokine ciliary neurotrophic factor (CNTF; n = 9) or 2) allowed to grow in culture media alone (n=7) . Each rat was injected with a total of 200,000 cells . A third group of rats (n=8) was given a sham injection of vehicle . Rats transplanted with human stem cells performed significantly better over the 8 weeks of testing on the cylinder test compared with those treated with vehicle (P < or = 0.001) . Stereological striatal volume analyses performed on Nissl-stained sections revealed that rats transplanted with CNTF-treated neurospheres had a 22% greater striatal volume on the lesioned side compared with those receiving transplants of untreated neurospheres (P = 0.0003) and a 26% greater striatal volume compared with rats injected with vehicle (P < or = 0.0001) . Numerous human nuclei-positive cells were visualized in the striatum in both transplantation groups . Grafted cells were also observed in the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata, areas of the basal ganglia receiving striatal projections . Some of the human nuclei-positive cells coexpressed glial fibrillary acidic protein and NeuN, suggesting that they had differentiated into neurons and astrocytes . Taken together, these data demonstrate that striatal transplants of human fetal stem cells elicit behavioral and anatomical recovery in a rodent model of Huntington's disease .

J Oral Rehabil, 2004 Jul, 31(7), 717 - 24
Changes in properties of short-term-use soft liners after thermocycling; Park SK et al.; The objectives of this study were to determine the influence of thermocycling on the changes of elastic modulus (EM) and colour, and to evaluate cytotoxicity after repeated elution of short-term-use soft liners . Three short-term-use soft liners {soft acrylic-based Coe Comfort (CCM), Coe Soft (CST) and Soft Liner (SFL)}, and long-term-use silicone-based Tokuso Soft Liner (TSL) acting as a control were studied . EM was measured at baseline and after thermocycling at 5-55 degrees C for 500, 1000, 1500 and 2000 cycles . For the colour measurement, specimens in discs 20 mm in diameter and 1 mm in thickness were prepared, attached to a denture base resin plate, and then thermocycled as above . Colour change (Delta E*) was measured according to the Commission Internationale de l'Eclairage (CIE) L*, a*, and b* scale on a spectrophotometer . For the cytotoxicity evaluation, specimens were eluted for 24 h in culture media repeatedly up to four times, and MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed . EM of CCM and CST increased up to 1500 and 1000 cycles of thermocycling respectively . EM of SFL gradually increased up to 2000 cycles, and that of TSL increased after 500 cycles and did not change after then . Delta E* of soft liners after 2000 cycles except CCM were 3.68-8.65 . EM increased up to 1000-1500 cycles, and perceivable colour change was observed after 2000 cycles in most materials . Therefore, short-term-use soft liners should be used within a limited time, although the cytotoxicity decreased after repeated elution.

Wei Sheng Yan Jiu, 2004 Mar, 33(2), 140 - 3
{Regulatory mechanism of EB1089 for hepatocarcinoma cell proliferation}; Luo W et al.; OBJECTIVE: Study regulatory mechanism of EB1089 for hepatocarcinoma cell proliferation . METHODS: HHCC 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 . The anti-proliferative effect were examined by MTT and the plate clone forming test . The apoptosis was detected by Electron Microscope and flow cell device . Westernblot were used to detect p27Kipl and PTEN expression . RESULTS: EB1089 could inhibit the proliferation of hepatocellular cell line HHCC . EB1089 could induce apoptosis of hepatocarcinoma cells . Western blot showed that EB1089 could elevate the expression level of p27Kipl and PTEN protein . CONCLUSION: EB1089's inhibition to hepatocarcinoma is probably realized through inducing apoptosis of hepatocarcinoma cells and elevating the expression level of p27Kipl and PTEN protein.

Fertil Steril, 2004 Jun, 81(6), 1657 - 64
Effect of Tisseel on expression of tissue plasminogen activator and plasminogen activator inhibitor-1; Diamond MP et al.; OBJECTIVE: To examine the effect of fibrin sealant on mRNA expression of factors regulating plasminogen activator activity in human peritoneal cells . Plasminogen activator activity is thought to play a pivotal role in degradation of the proteinaceous mass that develops after surgical procedures . Reduction of plasminogen activator activity, as occurs with tissue trauma, results in increased postoperative adhesion development . DESIGN: Tissue culture for 6, 12, 24, and 48 hours . SETTING: University research laboratory . PATIENTS: Source of mesothelial cells with fibroblasts . INTERVENTION(S): Measurement of mRNA expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) . MAIN OUTCOME MEASURE(S): Multiplex reverse transcriptase/polymerase chain reaction (RT/PCR) was used to determine relative change in t-PA and PAI-1 mRNA levels under six conditions: {1} . fibrin sealant (Tisseel); {2} . fibrin sealant (Tisseel) two components diluted 1:2; {3} . fibrin sealant (Tisseel) sealer protein component reconstituted without aprotinin (a protease inhibitor); {4} . fibrin sealant (Tisseel) sealer protein component reconstituted without aprotinin, both components diluted 1:2; {5} . fibrin sealant (Tisseel) components diluted to physiologic concentrations; and {6} control (culture media) . RESULTS: The mRNA levels of t-PA and PAI-1 by human peritoneal cells were unchanged during 48 hours . In mesothelial cells, the addition of the compositions increased t-PA mRNA levels . A selective increase was observed in the normal peritoneal fibroblasts at the later time points; similar increases were identified in adhesion fibroblast cultures . In mesothelial cells, the more concentrated compositions generally increased PAI-1 mRNA above control levels, whereas in normal peritoneal fibroblasts PAI-1 levels generally remained unchanged . In contrast, in adhesion fibroblasts, PAI-1 levels decreased over time with treatment . CONCLUSION(S): Fibrin sealant, in the presence and absence of aprotinin, increases both t-PA and PAI-1 expression by human peritoneal cells; changes not seen with physiologic concentrations of fibrin sealant . These observations suggest that in addition to its ability to help achieve hemostasis, fibrin sealant affects the healing process by altering components of the plasminogen activator system, which may be of benefit in the reduction of postoperative adhesions.

Fertil Steril, 2004 Jun, 81(6), 1502 - 6
Equivalency of culture media for human in vitro fertilization formulated to have the same pH under an atmosphere containing 5% or 6% carbon dioxide; Quinn P et al.; Culture media for human in vitro fertilization and early embryonic development were modified to maintain similar pH values under an atmosphere containing 5% or 6% carbon dioxide by adjusting the concentration of sodium bicarbonate . Similar results were obtained with both media groups in a mouse embryo assay and in human in vitro parameters of fertilization and early development and clinical outcomes.

Eur J Endocrinol, 2004 Jun, 150(6), 863 - 75
PPARgamma inhibits GH synthesis and secretion and increases apoptosis of pituitary GH-secreting adenomas; Bogazzi F et al.; OBJECTIVE: The objective of the study was to evaluate the expression and functional activity of Peroxisome proliferator-activated receptor (PPAR) gamma in pituitary adenomas from 14 consecutive acromegalic patients and to establish its role in apoptosis . SUBJECTS AND METHODS: Fourteen consecutive acromegalic patients were enrolled in the study . Wistar-Furth rats were used for in vivo studies . Expression of PPARgamma was evaluated by RT-PCR and Western blot . Apoptosis and cell cycle were assessed by FACS analysis . The effects of PPARgamma ligands on transcriptional regulation of GH gene were evaluated by RT-PCR and electromobility shift assay . RESULTS: PPARgamma was expressed in all human GH-secreting adenoma (GH-oma), in normal pituitary tissue samples (39+/-24% and 78+/-5% of immunostained nuclei respectively; P<0.0002; ANOVA), and in rat GH-secreting (GH3) cells . A PPRE-containing reporter plasmid transfected into GH3 cells was activated by ciglitazone or rosiglitazone (TZDs), indicating that PPARgamma was functionally active . Treatment of GH3 cells with TZDs increased apoptosis in a dose-dependent manner (P=0.0003) and arrested cell proliferation, reducing the number of cells in the S-phase (P<0.0001 vs untreated cells) . TZDs increased the expression of TRAIL, leaving unaffected that of p53 and Bax . TZDs reduced GH concentrations in the culture media from 43.7+/-5.4 ng/ml to 2.1+/-0.3 ng/ml (P<0.0001) and in cell extracts (P<0.004) . PPARgamma-RXRalpha heterodimers bound to GH promoter, inhibiting its activity and reducing GH mRNA levels (1.8 x 10(6) vs 5.7 x 10(6) transcripts respectively vs untreated cells; P<0.002) . Subcutaneous GH-oma developed in rats injected with GH3 cells; tumor growth increased in placebo-treated rats and to a lesser extent in TZDs-treated animals (24.1+/-2.0 g, and 14.8+/-4.2 g respectively, P<0.03) . Serum GH concentrations were lower in TZDs-treated rats than in controls (871+/-67 ng/ml vs 1.309+/-238 ng/ml; P<0.05) . CONCLUSIONS: The results of this study indicate that PPARgamma controls GH transcription and secretion as well as apoptosis and growth of GH-oma; thus, TZDs have the potential of a useful tool in the complex therapeutic management of acromegalic patients.

Cell Transplant, 2004, 13(3), 213 - 29
Comparison of bioenergetic activity of primary porcine hepatocytes cultured in four different media; Dabos KJ et al.; Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research . Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems . The optimum culture medium for hepatocytes to be used in such devices has yet to be defined . In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes . The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium . Cells (3 x 10(10); viability 87 +/- 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 x 10(6) viable cells per plate . Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis . ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9) . We also studied urea and albumin synthesis and total P450 content . The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them . All media performed well in glycolysis, ureagenesis, and albumin synthesis . William's E medium and medium 199 outperformed the rest in gluconeogenesis . Medium 199 was best in ketogenesis . Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.

Cell Transplant, 2004, 13(3), 197 - 211
Differentiation of human and mouse embryonic stem cells along a hepatocyte lineage; Shirahashi H et al.; Embryonic stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells . We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro . Various combinations of culture media, growth and differentiation factors, and substratum precoatings were evaluated, and it was determined that a combination of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone . and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage . Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver . The optimal culture condition also induced albumin gene expression in differentiated human ES cells 1% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocytochemistry . In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels 10-fold higher than with other hepatocyte differentiation media . In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage . This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.

Can J Vet Res, 2004 Apr, 68(2), 157 - 9
Use of a commercial methylcellulose medium with and without recombinant bovine granulocyte colony-stimulating factor for culturing bovine bone marrow cells; Keller SL et al.; A commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), was utilized for culturing bovine bone marrow cells in a colony-forming unit assay . Bone marrow mononuclear cells were isolated and cultured in a commercial methylcellulose-based medium containing several recombinant human cytokines . Cultures were prepared with and without 100 ng/mL of rbG-CSF . The size and mean number of colonies per plate from culture days 3 to 9 were compared . We concluded that bovine bone marrow colony growth was supported by this culture medium . The addition of rbG-CSF yielded larger and more numerous colonies . There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) with rbG-CSF . Both culture media had the highest colony counts on day 5.

Cancer Sci, 2004 Jun, 95(6), 547 - 52
Kigamicin D, a novel anticancer agent based on a new anti-austerity strategy targeting cancer cells' tolerance to nutrient starvation; Lu J et al.; Both tolerance to nutrient starvation and angiogenesis are essential for cancer progression because of the insufficient supply of nutrients to tumor tissue . Since chronic nutrient starvation seldom occurs in normal tissue, cancer's tolerance to nutrient starvation should provide a novel target for cancer therapy . In this study, we propose an anti-austerity strategy to exploit the ability of agents to eliminate cancer cells' tolerance to nutrient starvation . We established a simple screening method for agents that inhibit cancer cell viability preferentially during nutrient starvation, using PANC-1 cell line cultured in nutrient-rich and nutrient-deprived media . After screening over 2000 culture media of actinomycetes, we identified a new compound, kigamicin D (C(48)H(59)NO(19)), which shows preferential cytotoxicity to cancer cells under nutrient-deprived conditions, but hardly any cytotoxicity under nutrient-rich conditions . Both subcutaneous and oral administration of kigamicin D strongly suppressed the tumor growth of several tested pancreatic cancer cell lines in nude mice . Moreover, kigamicin D was observed to block the activation of Akt induced by nutrient starvation . Therefore, our results suggest that kigamicin D be a candidate for implementing our novel concept, anti-austerity, which may serve as a new strategy for cancer therapy.

In Vitro Cell Dev Biol Anim, 2004 Jan-Feb, 40(1-2), 14 - 21
Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice; Pei XF et al.; We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene-expressing transgenic mice and potentially other transgenic strains . By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo . Throughout the culture period, the explants produced abundant outgrowths of epithelial cells . As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another-a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells . This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.

Mycopathologia, 2004 Apr, 157(3), 269 - 71
Pulmonary aspergillosis outbreak in Rhea americana in southern Brazil; Copetti MV et al.; Commercial raising of rheas is currently in expansion in the south of Brazil, and many diseases previously restricted to other avian species are currently emerging on rhea farms, especially as a result of careless management of these animals . The objective of the present article is to report a pulmonary aspergillosis outbreak that occurred in great rhea (Rhea americana) in the south of Brazil . About 50 birds aged 30 to 60 days died suddenly and one of them was submitted to autopsy which revealed the presence of white caseous nodules 0.5 mm in diameter occupying 95% of the lung area . One lung was sent to the Federal University of Santa Maria for histopathological and mycological analyses . Histopathological analysis revealed multifocal areas with necrosis and inflammatory infiltrates and the presence of fungal hyphae, giant cells and fibrous tissue proliferation at the periphery . Aspergillus fumigatus was recovered as pure culture from all culture media . This appears to be the first report of aspergillosis among great rhea in Brazil and the second in the world.

Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 622 - 8
Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor; Yao Y et al.; In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist {(3)H}DCG-IV . The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined . Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding . The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507 . Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides . Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor . These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells.

Protein Expr Purif, 2004 Jul, 36(1), 61 - 9
High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer; Kotik M et al.; Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach . Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter . The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies . In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C . The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy . During the fed-batch process, lactose was added intermittently to the culture . A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained . Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium . Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O . In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.

Biotechnol Prog, 2004 May-Jun, 20(3), 897 - 904
Rapid protein anchoring into the membranes of Mammalian cells using oleyl chain and poly(ethylene glycol) derivatives; Kato K et al.; The cell membrane is an important interface for communication with extracellular events, and incorporation of bioactive substances, such as antibodies and receptors, into the cell membrane may enhance the potential abilities of cells . Gene manipulation, chemical modification of membrane proteins, and cell surface painting using a GPI anchor have been performed to introduce substances into cell membranes . Furthermore, many lipid anchors have also been used to modify lipid membranes such as liposomes . In this study, we have focused on developing an easy and rapid method for anchoring of substances including macromolecular proteins into the membranes of living mammalian cells . We employed a single oleyl chain derivative coupled with hydrophilic poly(ethylene glycol) (PEG90, the ethyleneoxide (EO) unit is 90) to facilitate solubilization in water . This water-soluble derivative was designated Biocompatible Anchor for Membrane (BAM) . Some proteins (streptavidin, EGFP and an antibody) were coupled with BAM90 at the distal terminal of PEG and rapidly (within a few minutes) anchored into the membranes of various cells (NIH3T3, 32D, Ba/F3, hybridoma 9E10) . However, the anchored BAM90 disappeared from the cell membranes within 4-5 h in serum-free culture media, and moreover, the retention time of anchoring was shortened (1-2 h) in culture medium containing 10% FBS . We further prepared a dioleylphosphatidylethanolamine (DOPE)-PEG derivative (DOPE-BAM80, the EO unit is 80) as a double oleyl chain derivative for comparison with the single oleyl chain derivative, BAM90 . The retention time of anchored DOPE-BAM80 was longer than that of BAM90 and more than 8 h in culture media with and without 10% serum . Furthermore, the treatment time of DOPE-BAM80 for anchoring was nearly as short (within a few minutes) as that of BAM90 . In addition, both types of BAMs, BAM90 and DOPE-BAM80, showed no cytotoxicity . Therefore, DOPE-BAM80 is useful for protein anchoring to cells . Although the utilization of BAM90 is considered to be limited, it is expected to useful in restricted environments, for example, in tissues such as the cornea, peritoneum, bladder, and various mucosae, which are less exposed to serum . Thus, we suggest the possibility that both types of BAM can be applied to cell surface engineering.

Prostate, 2004 Aug 1, 60(3), 175 - 7
Endothelin A receptor blockade does not alter PSA secretion in prostate cancer cell lines; Pecher S et al.; BACKGROUND: Some men treated with atrasentan (ABT-627), an endothelin A (ETA) receptor inhibitor, had declines in their serum PSA levels . It is our hypothesis that this decrease is due to anti-tumoral activity and not a reduction in PSA secretion at the cellular level . METHODS: Two PSA secreting prostate cancer cell lines (LAPC4 and LNCaP) were treated with atrasentan and an ETB receptor antagonist (A192621) in varying concentrations (10(-6)-10(-10) M) and PSA levels were measured in the culture media . RESULTS: LNCaP and LAPC4 cells both express ETA receptors . Neither the ETA or ETB antagonist altered PSA secretion, while addition of DHT, a positive control, produced a marked increase in PSA secretion . CONCLUSIONS: Blockade of the ETA receptor does not affect the secretion of PSA in prostate cancer cell lines .

Biol Res, 2004, 37(1), 45 - 51
Optimization of a protocol for direct organogenesis of red clover (Trifolium pratense L.) meristems for breeding purposes; Carrillo JC et al.; A series of experiments were carried out in order to optimize a protocol for the direct organogenesis of Chilean red clover germplasm . A range of cultivars were used to analyze the effect of explant source (crown or stem meristems of vegetative plants), culture media and plant growth regulators . Our findings showed that stem meristems were easier to obtain, presented lower levels of contamination and a better development than crown meristems . The L2 medium showed better results than B5 and MS media for the cultivars and experimental lines studied . L2 medium supplemented with 0.003 mg/l of 4-amino-3,5,6-trichloropicolinic acid and 1.0 mg/l of 6-benzylaminopurine gave consistently better results and will be applied in our breeding program to propagate, maintain and eliminate viruses from elite red clover clones.

J Nutr, 2004 Jun, 134(6), 1493 - 9
Prenatal protein restriction does not affect the proliferation and differentiation of rat preadipocytes; Bieswal F et al.; Poor development in utero may favor the development of obesity in adulthood . Animal studies showed that embryo manipulation in vitro or nutritional insults during the embryonic and fetal stages of development may lead to obesity in adult life . We studied the in vitro proliferation and differentiation of adipocytes to investigate whether early protein restriction may program cell growth and development . In a series of experiments, 2 different low-protein diet protocols were compared . In both cases, pregnant rats were fed a diet with a high (18-20%) or low (8-9%) protein content during gestation and/or lactation . Preadipocytes were isolated from the fetuses, neonates, and weanling offspring . Moderate protein restriction, imposed during either gestation and/or lactation, did not affect the capacity of preadipose cells to divide or store fat . Because previous studies showed that early protein restriction alters the metabolism of sulfur amino acids, we also investigated the effects of methionine, taurine, and homocysteine on proliferation and differentiation of preadipocytes . The supplementation of the diet with methionine or the addition of homocysteine and taurine to the culture media did not influence the development of preadipocytes . We obtained no evidence for the direct reprogramming of the precursor or stem cells and suggest that the subsequent alteration in fat accretion may therefore reflect a change in the neuroendocrine environment.

Obstet Gynecol, 2004 Jun, 103(6), 1154 - 63
Are children born after assisted reproductive technology at increased risk for adverse health outcomes?
Schieve LA, Rasmussen SA, Buck GM, Schendel DE, Reynolds MA, Wright VC.
As assisted reproductive technologies (ARTs) are increasingly used to overcome infertility, there is concern about the health of the children conceived . The empirical evidence for associations with outcomes related to child health is variable and should be evaluated with consideration of methodological shortcomings . Currently, there is convincing evidence that ART treatment may increase the risk of a few outcomes . Experimental laboratory studies document that various constituents in culture media affect various embryo characteristics both positively and negatively . Multiple-gestation pregnancy and birth are increased with ART, both because of multiple embryo transfer and embryo splitting . There is evidence of an increase in chromosomal abnormalities among pregnancies conceived using intracytoplasmic sperm injection and low birth weight and preterm delivery among singletons conceived with all types of ART; however, there remains uncertainty about whether these risks stem from the treatment or the parental infertility . For some outcomes, data of an increased risk with ART are suggestive at best largely because of lack of purposeful study of sufficient size and scope . These include specific perinatal morbidities, birth defects, developmental disabilities, and retinoblastoma . The evidence for an association between ART and spontaneous abortion is inconsistent and weak . There is inconclusive evidence that ART may be associated with genetic imprinting disorders . For childhood cancer, chronic conditions, learning and behavioral disorders, and reproductive effects there is insufficient empirical research to date, but given the data for more proximal outcomes, these outcomes merit further study . Future research needs to address the unique methodological challenges underlying study in this area.

Curr Microbiol, 2004 Jun, 48(6), 391 - 5
Regulation of the htpX gene of Xylella fastidiosa and its expression in E . coli; Coltri PP et al.; Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies . In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions . This gene was induced after a temperature upshift to 37 degrees C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts . Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E . coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37 degrees C or 45 degrees C in all culture media tested, but not at 28 degrees C in minimal culture medium . The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting.

Drug Deliv, 2004 Jan-Feb, 11(1), 11 - 8
Transepithelial electrical resistance is not a reliable measurement of the Caco-2 monolayer integrity in Transwell; Mukherjee T et al.; The significance of monitoring transepithelial electrical resistance (TEER) value during the study on drug absorption through Caco-2 monolayers in Transwells was re-evaluated . TEER value was monitored before, during, and after the absorption of Streptokinase (45 KD) . Four enhancers--disodium ethylenediaminetetracetate (disodium EDTA), sodium cholate (NaC), sodium taurocholate (NaTC), and sodium caprate along with alpha-hemolysin (a cell membrane pore-forming toxin)--were used to signify the outcome of this study . Modified trypan blue exclusion technique was used to examine the Caco-2 cell viability throughout the absorption studies . The enhancers at the used concentration exhibited toxic effect on the Caco-2 cells as evident from the trypan blue exclusion studies . This toxic effect was not reflected by the TEER profile because TEER value dropped after the addition of the absorption enhancers . But it came back to its initial value after the cell culture media was replaced by enhancer-free media . This toxic effect was confirmed by the antiproliferation studies on the four enhancers and alpha-hemolysin against Caco-2 cells . Therefore, we concluded that the measurement of TEER is not a reliable method to determine the absorption enhancers toxicity or integrity of the Caco-2 monolayers in the Transwells.

Hum Reprod, 2004 Aug, 19(8), 1821 - 5 Epub 2004 May 27.
A combination of interleukin-6 and its soluble receptor impairs sperm motility: implications in infertility associated with endometriosis; Yoshida S et al.; BACKGROUND: We previously reported that the level of interleukin (IL)-6 is increased in the peritoneal fluid of women with endometriosis . This study was undertaken to assess the effects of IL-6 and soluble IL-6 receptor (sIL-6R) on in vitro sperm motility . METHODS: Sperm (n = 20) were cultured with IL-6 or sIL-6R, or with a combination of both . After 24 h cultures, sperm motility was evaluated using a computer-assisted semen analysis system . Gene and protein expressions of IL-6, IL-6 receptor (IL-6R), and glycoprotein 130 (gp130) were examined in sperm by RT-PCR analysis and western blot analysis . RESULTS: Addition of IL-6 or sIL-6R individually to the culture media had no affect on sperm motion . However, adding a combination of IL-6 and sIL-6R dose-dependently reduced the percentage of motile and rapidly moving sperm . Adding anti-IL-6R antibody abolished these adverse effects . Sperm expressed the gp130 gene and protein, but not IL-6 or IL-6R . CONCLUSIONS: A combination of IL-6 and sIL-6R may be associated with gp130 expressed in the sperm and reduce sperm motility . IL-6 and sIL-6R may contribute to the pathogenesis of endometriosis-associated infertility .

Metabolism, 2004 Jun, 53(6), 766 - 71
Effect of glucosamine on apolipoprotein AI mRNA stabilization and expression in HepG2 cells; Haas MJ et al.; Previously published studies suggest that an alteration in hexosamine flux induces a state of insulin resistance in muscle, liver, and other cell types . Glucosamine also alters the expression of several genes through an effect on transcription factors such as Sp1 . Since the anti-atherogenic protein apolipoprotein AI (apoAI) is positively regulated by insulin, at least partly through its effect on Sp1, we investigated the effect of glucosamine on apoAI gene expression in the hepatocyte cell line, HepG2 . By 24 hours of treatment with 0.1, 1, or 3 mmol/L glucosamine, the amount of apoAI protein secreted into the culture media increased 1.8-fold, 5.5-fold, and 2.3-fold, respectively . The decline in apoAI secretion at the highest glucosamine levels may be due to toxicity since the percentage of cells able to exclude trypan blue was lower in this group than in control cells (98.5% +/- 1.5% in control cells v 89.2% +/- 2.1% in cells treated with 3 mmol/L glucosamine, P <.01) . ApoAI mRNA levels increased 2.4-fold in hepatocytes treated with 1 mmol/L glucosamine for 24 hours (1,158.1 +/- 78.8 v 482.2 +/- 24.3 arbitrary integrator units {AIU}, P <.02), suggesting that the increase in apoAI protein secretion was due, at least partly, to an increase in apoAI mRNA levels . However, glucosamine had no effect on apoAI gene transcription rate as measured by nuclear runoff analysis (3,155 +/- 46.0 in control cells v 3,181 +/- 30.0 AIU in glucosamine-treated cells) . Similarly, apoAI promoter activity measured in HepG2 cell transfected with an apoAI reporter plasmid containing the full-length apoAI promoter including an insulin-responsive Sp1 binding site did not change with glucosamine addition . In this assay, the chloramphenicol acetyltransferase (CAT) activity was 12.4% +/- 3.1%, 10.1% +/- 2.4%, 9.8% +/- 2.0%, 9.7% +/- 2.2%, and 11.9% +/- 2.9% in cells treated with 0, 0.03, 0.1, 0.3, and 1 mmol/L glucosamine, respectively . The apoAI mRNA turnover studies showed that 1 mmol/L glucosamine treatment of HepG2 cells was associated with increased apoAI mRNA half-life, from 7.6 to 16.6 hours . These findings suggest that increases in apoAI gene expression by glucosamine occur primarily through stabilizing apoAI mRNA.

Vaccine, 2004 Feb 25, 22(8), 1032 - 46
Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1; Jeffs SA et al.; The production, purification and characterisation of recombinant gp140 oligomeric envelope glycoproteins derived from six primary isolates of HIV-1 (covering clades A, B, C, D, F and O) are described . Using a Chinese hamster ovary cell expression system, expression levels of between 0.1 and 1 mg/l cell-conditioned culture media were obtained, and purified to >95% by affinity chromatography . A, B, D, F and O clade gp 140s were found to be multimeric, bind to a panel of defined env-specific monoclonal antibodies and interact with CD4 and CXCR4, demonstrating correct folding . Their immunogenicity was confirmed by the generation of high-titre anti-gp140 antibodies in rabbits . The C clade gp140 was incorrectly folded and poorly antigenic . Despite the presence of an unmodified gp120/41 cleavage site, only the B clade gp140 showed significant processing to gp120 and gp41 . Each gp140 has a specific pattern of oligomerisation, and varies in its resistance to reducing agents and salt concentration . The binding of gp140 to soluble and cell-surface CD4 and CXCR4 is related to the degree of oligomerisation . The C1 and C5 regions, CD4 binding domain and the epitope defined by the 2G12 monoclonal antibody were well exposed, but the C-terminal region of the extracellular domain of gp41 appears to be occluded by oligomerisation . These reagents have potential as immunogens for use in vaccine development.

Neurosci Lett, 2004 May 27, 362(3), 220 - 5
Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis; Davis RL et al.; Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma . Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment . However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown . We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 microm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 microg/ml lipopolysaccharide (LPS) + 10 ng/ml interleukin (IL)-1beta-stimulated A172 human astroglia cells . The involvement of the chemokine CXCL10 (also known as interferon-gamma inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody . Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS + IL-1beta . Furthermore, we observed that LPS + IL-1beta-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol . Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies .

Int J Parasitol, 2004 Jun, 34(7), 779 - 84
Trypanosoma cruzi: long-term sub-cultures in two different culture media do not confirm the existence of highly versatile multilocus genotypes; Barnabe C et al.; Trypanosoma cruzi Y reference strain is found in many laboratories under at least two highly distinct genotypes, A and B corresponding to the 'discrete typing units' T . cruzi IIb and T . cruzi IId, respectively . Previous work has reported reversible switches between these genotypes according to the culture media used in the experiments: genotype A would be associated with blood-enriched culture media, while genotype B would be associated with blood-free culture media . We tried to reproduce this observation, but used a different cloning method of individual organisms . Our cloning was verified visually under the microscope, while the previous studies relied on a cloning by dilution only . The subclones so obtained were submitted to long-term exposure to both media, and no change was observed in isoenzyme and random amplified polymorphic DNA genotypes . The discrepancy is probably explained by the cloning method: clones obtained from the previous method (dilution and plating) could come from several parasite cells while only one cell generates a clone when micro-manipulation is used.

AIDS Res Hum Retroviruses, 2004 Apr, 20(4), 383 - 97
When integrated in a subepithelial mucosal layer equivalent, dendritic cells keep their immature stage and their ability to replicate type R5 HIV type 1 strains in the absence of T cell subsets; Dumont S et al.; Many potential targets of human immunodeficiency virus type 1 (HIV-1) reside in the human reproductive tract, including dendritic cells (DC) . The ability of these cells to replicate HIV-1 is dependent on many factors such as their differentiation/maturation stage . Nevertheless, precise mechanisms underlying the early steps of transmucosal infection are still unknown . Our purpose was to investigate DC/HIV-1 interactions in a subepithelial mucosal layer equivalent (SEMLE) reconstructed in vitro . We used mixed interstitial DC (IntDC)/Langerhans cell (LC)-like cell subpopulations generated in vitro from CD34(+) progenitors . These cells were either integrated in SEMLE or maintained in suspension . Experimental infections were performed with a type X4 strain (HIV-1(LAI)) and a type R5 strain (HIV-1(Ba-L)) . Proviral DNA was detected by in situ polymerase chain reaction (PCR) and viral replication was quantified by measuring p24 core protein release in the culture media . Our results showed that SEMLE enable DC to retain immature stage and reproduce the tropic selection that occurs in vivo . Indeed, IntDC/LC were infected by both types of HIV-1 strains, regardless of the infection schedule, whereas only type R5 virus replicated in DC in the absence of T cell subsets . Furthermore, the ability of DC to replicate HIV-1(BaL) was lost after 14 days of culture unless the cells had previously been integrated in SEMLE . These results suggest that this 3D model maintains the ability of DC to replicate type R5 virus by delaying their maturation . In conclusion, this in vitro model mimics human submucosa and can be considered as relevant for studying the preliminary steps of transmucosal HIV-1 infection.

Appl Microbiol Biotechnol . 2004 May 20; {Epub ahead of print}
Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity; Suarez B et al.; Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism . Several proteins with proteolytic activity were detected in culture filtrates from T . harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity . The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7-4.9 . The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity . Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1 . The protein sequence showed <43% identity to mammal trypsins and 47-57% to other fungal trypsin-like proteins described thus far . Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media . The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations . This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T . harzianum CECT 2413 . A role for PRA1 in the protection of plants against pests and pathogens provided by T . harzianum CECT 2413 is proposed.

Methods Mol Biol, 2004, 268, 133 - 7
Determination of aflatoxins and zearalenone in different culture media; Bueno DJ et al.; Some molds produce desirable changes in food, but most are merely esthetically undesirable . There has also been an increasing awareness that certain metabolic products of some molds commonly found on foods and feed are dangerous to humans and animals . These toxin substances, mycotoxins, are secondary metabolites produced by different fungi, especially Aspergillus, Penicillium, Fusarium, and, to a lesser degree, Alternaria . The most important toxins for humans are aflatoxins, ochratoxin A, fumonisins, certain trichothecenes, and zearalenone.Aflatoxins are fungal metabolites produced by different Aspergillus species: A . flavus, A . parasiticus, and A . nomius . The most commonly encountered aflatoxins are B1, B2, G1, G2, M1, and M2, but aflatoxin B1 is the most frequently found in contaminated samples, and aflatoxins B2, G1, and G2 are generally not reported in the absence of AFB1 . The International Agency for Research on Cancer (IARC) considers that aflatoxins are carcinogenic (hepatocarcinogenic) to humans (group 1) and animals.Zearalenone is an estrogenic mycotoxin produced by several species of Fusarium (F . acuminatum, F . culmorum, F . equiseti, F . graminearum, F . verticilliodes, F . oxysporum, F . poae, F . rosum, F . solani, F . semitectum, and F . sporotrichioides) that primarily colonize different cereal grains . Several reports were noted on the occurrence of ZEA along with various combinations of group B trichothecenes, fumonisins, aflatoxins, and ochratoxins . In most cases, the levels of ZEA were considered to be low; however, the toxicological significance is not known . This toxin is not classifiable as to carcinogenicity in humans (group 3) by the IARC, but ZEA was implied in precocious sexual development in children in Puerto Rico and a breast enlargement in young boys in Italy.

J Pharmacol Sci, 2004 May, 95(1), 65 - 70
CD14 glycoprotein expressed in vascular smooth muscle cells; Choi HC et al.; The inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells is an important factor for pathogenesis of septic shock or multiple organ dysfunction syndrome . The mechanisms of iNOS expression in such conditions are partly known . This study tried to clarify the signal transduction of lipopolysaccharide (LPS) single stimulation that induces iNOS mRNA and protein in vascular smooth muscle cells (VSMC) . VSMC were primarily cultured from rat aorta . The concentrations of nitrite in culture media were measured by the Griess reaction . Western blottings and immunoreaction for iNOS, nuclear factor kappaB (NFkappaB) p65, and CD14 protein were performed . mRNAs of iNOS and tumor necrosis factor (TNF) alpha were analyzed by RT-PCR . Genistein inhibited LPS induced early phase nitrite production, while pyrrolidine dithiocarbamate (PDTC) inhibited nitrite production at a late phase . PDTC significantly reduced NFkappaB p65 and iNOS protein expression by LPS . TNFalpha mRNA expression by LPS was not detected in VSMC . Membranous CD14 glycoprotein was detected in VSMC and soluble CD14 glycoprotein was not detected in fetal bovine serum added in culture media . These results suggest that CD14 glycoprotein is present on the cell membranes of VSMC, a non-myelomonocyte lineage, acting as an LPS receptor . Activations of tyrosine kinase and NFkappaB p65 are essential for iNOS expression by LPS single stimulation, while TNFalpha is not a concern to iNOS expression in VSMC.

Biol Reprod, 2004 Sep, 71(3), 948 - 58 Epub 2004 May 19.
Postthaw evaluation of in vitro function of epididymal spermatozoa from four species of free-ranging African bovids; Herrick JR et al.; An improved understanding of reproductive physiology in nondomestic bovids is necessary for the development of assisted reproductive technologies (ARTs) for use in the conservation of endangered bovids . In this study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls in South Africa . Our objectives were 1) to characterize the quality of epididymal spermatozoa, 2) evaluate the effectiveness of a cryopreservation protocol, and 3) compare postthaw sperm longevity (motility, viability, and acrosomal integrity) and functionality in two culture media with two capacitation reagents (caffeine and heparin) . Following recovery, spermatozoa were diluted in EQ extender, slow-cooled, and frozen in the presence of 5% glycerol . Thawed spermatozoa were separated on a Percoll gradient and diluted in fertilization media (SOF for fertilization {SOFfert}; 0.6% BSA, 0.0 mM glucose, 25.0 mM NaHCO(3)) or modified SOFfert (1.2% BSA, 1.5 mM glucose, 37.0 mM NaHCO(3)) and either heparin or caffeine, and incubated for 6 h . Spermatozoa from these species maintained an average of 64% initial motility after thawing . Incubation medium and capacitation reagent had species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa, suggesting ART procedures need to be optimized for each species . Springbok spermatozoa were also shown to be competent for in vitro fertilization . Information from this study concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest will be useful in the development of ART for the conservation of these and other species of bovids.

Int J Biochem Cell Biol, 2004 Aug, 36(8), 1635 - 44
Purification and characterization of decorin from the culture media of MRC-5 cells; Honda E et al.; Myofibroblasts play an important role in fibrogenesis . Myofibroblasts secrete several components of the extracellular matrix, including decorin . To clarify the properties of decorin synthesized by myofibroblasts, we have purified and characterized decorin secreted into culture medium by the myofibroblast cell line MRC-5 . Decorin was purified by successive chromatography steps using Hitrap Q and Superdex 200 . Purified decorin showed a broad band on SDS-polyacrylamide gel electrophoresis, which was resolved into two smaller molecular weight bands after digestion with chondroitinase ABC . Further digestion with N-glycanase resolved these two bands into a single band, indicating that the N-glycation pattern of decorin is heterogeneous . The N-terminal amino acid sequence analysis of the purified protein and its reactivity towards an antibody raised against a C-terminal peptide of decorin indicate that MRC-5 cells secrete full-length decorin into the culture medium . To characterize the glycosaminoglycan chains attached to decorin, glycosaminoglycans from the purified protein were treated with chondroitinase ACI, chondroitinase ACII, chondroitinase ABC and chondroitinase B . The resulting disaccharides were analyzed by chromatography, which indicated that decorin secreted by MRC-5 cells is a dermatan sulfate proteoglycan . In conclusion, the decorin secreted by MRC-5 cells has similar characteristics to the decorin expressed in several tissues . Thus, culturing MRC-5 cells may be highly useful for studying the role of decorin and myofibroblasts in fibrosis.

Int J Biochem Cell Biol, 2004 Aug, 36(8), 1462 - 72
Kupffer cell cytokines interleukin-1beta and interleukin-10 combine to inhibit phosphoenolpyruvate carboxykinase and gluconeogenesis in cultured hepatocytes; Yerkovich ST et al.; BACKGROUND AND AIMS: Recent evidence suggests that inflammatory cytokines may mediate reduced hepatic glucose production and reduced blood glucose concentrations in sepsis . Therefore the aim of this study is to provide direct evidence of a cytokine-mediated interaction between Kupffer cells and hepatocytes by characterising the effects of lipopolysaccharide-stimulated Kupffer cells on hepatocyte gluconeogenesis, and the activity of key regulatory enzymes of this pathway . METHODS AND RESULTS: Primary isolates of hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells in Transwell inserts showed a 48% inhibition of gluconeogenesis (P < 0.001) . RNase protection assay and ELISA of Kupffer cells and the culture media following exposure to lipopolysaccharide showed increased levels of interleukin-1 alpha and beta, tumour necrosis factor alpha and IL-10 . The addition of IL-1beta and IL-10 to hepatocyte cultures inhibited gluconeogenesis by 52% (P < 0.001), whereas each cytokine alone was ineffective . To determine whether altered production or activity of phosphoenolpyruvate carboxykinase or pyruvate kinase was responsible for the reduced glucose synthesis, their mRNA, protein levels and enzyme activities were measured . Primary hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells or cultured with a combination of IL-1beta and IL-10 displayed reduced levels of phosphoenolpyruvate carboxykinase mRNA, protein and enzyme activity . In contrast the mRNA, protein levels and enzyme activity of pyruvate kinase were not altered; suggesting that gluconeogenesis was suppressed by downregulation of phosphoenolpyruvate carboxykinase . CONCLUSIONS: Therefore, hypoglycaemia, which is often observed in sepsis, may be mediated by Kupffer cell-derived IL-1beta and IL-10 . In addition this study suggests these cytokines inhibit phosphoenolpyruvate carboxykinase production and thereby hepatic gluconeogenesis.

Endocr Regul, 2004 Mar, 38(1), 15 - 21
Estrus cycle-dependent action of leptin on basal and GH or IGF-I stimulated steroid secretion by whole porcine follicles; Gregoraszczuk EL et al.; OBJECTIVE: To determine the levels of leptin in the follicular fluid and using culture of whole ovarian follicles, to test the hypothesis that leptin may directly influence GH and IGF-I stimulated ovarian function . METHODS: Porcine follicles were recovered from ovaries during early, middle, and preovulatory stage of the follicular phase of the estrus cycle . They were cultured in the presence of the recombinant ovine leptin (oLEP) added either alone or with oGH or hIGF-I . Steroid concentrations in the media were determined after 48 h of culture . RESULTS: The respective values for leptin in follicular fluid from small, medium and large follicles were 1.98, 2.18 and 1.96 ng/ml, respectively . Leptin added alone at a dose of 2 ng/ml had no effect on basal steroid secretion by small and medium follicles . However, in small follicles a synergic action of GH and IGF-I was noted . Leptin did not influence the secretion of progesterone by follicles collected during the early and middle follicular phases . In preovulatory follicles, leptin added alone to the culture media caused a decrease in basal estradiol secretion with a concomitant increase in progesterone secretion . Moreover, it acted synergistically with IGF-I and GH causing further stimulation of progesterone secretion . CONCLUSIONS: The presented data show a direct, maturation dependent action of leptin on GH and IGF-I stimulated follicular steroidogenesis . During follicular growth they acted synergistically with GH and IGF-I in estradiol production, while in preovulatory follicles, they acted with both investigated hormones in luteinization process, which starts before follicular disruption.

J Biomater Sci Polym Ed, 2004, 15(3), 357 - 70
Competitive plasma protein adsorption on modified polymer surfaces monitored by quartz crystal microbalance technique; Welle A; This paper describes the effects of photochemical modifications of polymer surfaces on the competitive adsorption of serum proteins and cell adhesion (hepatoma cell line HepG2, L929 fibroblasts and others) . The UV modification of polystyrene, poly(methylmethacrylate) and polycarbonate alters the physico-chemical properties of these polymers in a way that allows the formation of micrometer scaled cellular patterns in vitro by controlling the composition and properties of the protein adsorbate . Using a quartz microbalance technique, capable to extract viscoelastic data in addition to the mass load of the polymer coated sensor, we have demonstrated the importance of the thickness and the viscosity of an albumin adsorbate for the observed cell adhesion in vitro . The quantity and viscosity of surface bound albumin on polystyrene, being a cell repellent material in its native state, is lowered when the surface is exposed to UV of lambda = 185 nm in air prior to the contact with albumin solutions or cell culture media . This promotes the deposition of cell adhesion proteins and explains the observed cell patterns . Apart from this special application the described quartz microbalance with dissipation monitoring provides a useful tool for general biocompatibility studies based on surface phenomena of biomaterials.

Int J Food Microbiol, 2004 May 1, 92(3), 255 - 63
Virulence and risk from drinking water of heterotrophic plate count bacteria in human population groups; Edberg SC et al.; Bacteria are natural inhabitants of all aqueous environments . The heterotrophic plate count is a means of assessing the concentration of these bacteria in foods, water, and water filtration systems . Methods vary, but are designed to enumerate bacteria that have evolved an environmental lifestyle . Most commonly, low nutrient, low ionic strength culture media are employed . The group of environmental bacteria enumerated depends on the media formulation and incubation conditions but are commonly known as heterotrophic plate count (HPC) bacteria; in Europe, this group is also referred to as autochthonous flora . While HPC inhabit an environmental niche, there has been concern that at some concentration they may be a human health risk . A review of the literature, including animal and human feeding studies, analysis of virulence factors, and outbreaks demonstrates that HPC bacteria as enumerated on HPC culture media have not been established as a human health threat at any concentration in drinking water or foods .

Zhonghua Yi Xue Za Zhi, 2004 Apr 2, 84(7), 587 - 91
{Effects of anti-sense Smad4 gene on the biological characteristics of the fat-storing cell line CFSC}; Xu XB et al.; OBJECTIVE: To investigate the effects of antisense Smad(4) on the biological characteristics of the fat-storing cell line CFSC . METHODS: Fat-storing cells of line CFSC from rat with liver fibrosis were cultured and transfected with 50 MOI of recombinant adenoviral vector carrying antisense Smad(4) (AdvATSmad(4)) or the control empty adenovirus (Adv0), both produced by 293 packaging cells, respectively . Two, four, and six days after the transfection the cultured cells were collected to undergo trypan blue staining and cell counting . The growth curves were drawn . The presence of antisense Smad(4) was detected by RT-PCR and Western blotting . (3)H-TdR was added into the culture media to be co-cultured for 6 hours . Then the cells were collected to examine the (3)H-TdR incorporation rate . RT-PCR and immunohistochemistry were used to examine the expression of COL1A1 and type I collagen, kinds of extracellular matrix (ECM) . RESULTS: Compared with the control CFSC and the Adv0-transfected CFSC cells, the cell growth curve, (3)H-TdR incorporation rate, proline incorporation rate, expression of Smad(4), and expression of extracellular matrix were markedly decreased in the AdvATSmad(4)-transfected CFSCs . CONCLUSION: The antisense Smad(4) gene inhibits the expression of Smad(4) mRNA and protein, proline incorporation and cell growth, thus down-regulating the production of ECM . Antisense Smad(4) gene may be used as a choice of gene therapy for liver fibrosis.

Clin Exp Allergy, 2004 May, 34(5), 704 - 11
Human airway submucosal glands augment eosinophil chemotaxis during rhinovirus infection; Furukawa E et al.; BACKGROUND: Asthma exacerbations are frequently associated with rhinovirus (RV) infections . However, the contribution of airway submucosal gland (SMG) to exacerbations of asthma in RV respiratory infection has not been studied . OBJECTIVE: This study was undertaken to examine whether RV-infected human respiratory SMG cells produce pro-inflammatory cytokines and chemokines for eosinophils, and augment eosinophil transmigration across human airway epithelium . METHODS: We infected cultured human tracheal SMG cells with RV14, collected culture media at 1, 3, and 5 days after infection, and measured the chemotactic activity for eosinophils in the culture supernatant using a 48-well microchemotaxis chamber and a (51)Cr-labelled eosinophil transmigration assay . RESULTS: Exposing a confluent human tracheal SMG cell monolayer to RV14 consistently led to infection . Human SMG cells with RV infection secreted soluble factors activating human eosinophil chemotaxis into the culture supernatant in a time-dependent manner, and the culture supernatant significantly augmented the transmigration of (51)Cr-labelled eosinophils through human airway epithelial cell layers from the basal to mucosal side . These effects were completely abolished by a mixture of a monoclonal antibody regulated on activation, normal T cells expressed and secreted (RANTES) and an antibody to granulocyte macrophage-colony stimulating factor (GM-CSF) . CONCLUSION: These results suggest that human respiratory SMG cells may augment eosinophil transmigration across the airway epithelium through the secretion of RANTES and GM-CSF after RV infection, and may contribute to exacerbations of asthma.

Am J Vet Res, 2004 May, 65(5), 604 - 9
Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage; Cook JL et al.; OBJECTIVE: To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage . SAMPLE POPULATION: Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers . PROCEDURE: Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency . Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured . Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores . Liquid culture media were collected every 4 days and GAG content measured . Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37 degrees, 45 degrees, or 55 degrees C . Cell viability was determined via 2 different assays immediately and 24 hours after treatment . RESULTS: Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining . Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period . Cell viability was significantly different between controls and cells treated at 55 degrees C immediately and 24 hours after heat treatment . CONCLUSIONS AND CLINICAL RELEVANCE: In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences . The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation.

Reprod Nutr Dev, 2003 Nov-Dec, 43(6), 487 - 96
Macromolecular source as dependent on osmotic pressure and water source: effects on bovine in vitro embryo development and quality; Duque P et al.; This study evaluated the protective effect of protein, as dependent on osmolarity, and the quality of water sources used to prepare embryo culture media . In Experiment 1, two concentrations of NaCl were used to obtain culture media with normal (280 mOSM) and low (245 mOSM) osmolarity, each supplemented with either bovine serum albumin (BSA) or polyvinyl alcohol (PVA) . Low osmolarity improved blastocyst rates in the presence of BSA (P < 0.01) and tended to do it in medium containing PVA (P < 0.07) . Furthermore, low osmolarity allowed PVA to increase inner cell mass (ICM) numbers and ICM/total cell rate (P < 0.05), while trophectoderm (TE) and total cell counts tended to decrease (P < 0.08) . In Experiment 2, culture media were prepared with two water sources (Milli-Q and Sigma-W3500-) in combination with BSA or PVA . Both water sources yielded similar embryo development rates, but in the presence of BSA, Milli-Q water produced embryos with increased ICM/total cells rates (P < 0.05) . On the contrary, Sigma water tended to increase trophectoderm cell counts (P < 0.08) . In conclusion, the present study showed that low osmolarity is beneficial to embryo development and combinations of macromolecule and osmolarity influence trophectoderm differentiation . Both Milli-Q and Sigma supported embryo development at comparable rates, although in the presence of BSA, blastocysts obtained in the medium prepared with Milli-Q water had superior quality in terms of ICM/total cells rates.

Histochem Cell Biol, 2004 May, 121(5), 407 - 18 Epub 2004 May 12.
Dynamics of polymorphism of acidocalcisomes in Leishmania parasites; Miranda K et al.; Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes . When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 microm diameter distributed throughout the cell . X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc . Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes . When cells were transferred to Warren's medium, the acidocalcisomes transformed from spherical into branched tubular organelles . The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes . X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron . Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.

Protein Expr Purif, 2004 Jun, 35(2), 218 - 24
Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms; Meier TI et al.; The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides . Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct . To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells . None of the p110 subunits were successfully expressed in the absence of p85alpha . Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells . Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient . The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta . The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting . The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome . All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2 . The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta . The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms . These kinetic differences may contribute to the unique roles of these isoforms in cells.

Osteoarthritis Cartilage, 2004 Jun, 12(6), 497 - 505
Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts; Yates KE; OBJECTIVE: The Wnt family of secreted proteins, their receptors (Fzd proteins) and antagonists (secreted Fzd-related proteins, or Sfrp) regulate chondrocyte differentiation and chrondrogenesis during embryonic development . Here, the hypothesis that the Wnt regulatory network contributes to chondrocyte differentiation of post-natal cells was tested in an in vitro model of chondroinduction by demineralized bone powder (DBP) . DESIGN: Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP . In some experiments, lithium chloride (LiCl), an agonist of the Wnt/beta-catenin signaling pathway, was added to the culture media . Sponges were cultured for intervals (0.5-21 days) before processing for molecular, histologic, and biochemical analyses . Expression of wnt, fzd, and sfrp genes was characterized by semi-quantitative RT-PCR . Fibroblasts' contacts with DBP were documented by histology . Accumulation of proteoglycan in extracellular matrix was evaluated by histology (metachromasia in toluidine blue-stained sections) and quantitative immunoassay (chondroitin 4-sulfate ELISA) . RESULTS: Expression of 15 wnt, fzd, and sfrp family members was detected in hDFs by RT-PCR . A subset of those genes (wnt2b, wnt5b, wnt10b, fzd6, fzd7) showed altered expression in hDFs exposed to DBP for 3 days . wnt and fzd gene expression was not altered before hDFs contacted the DBP within the collagen sponge . Human DFs cultured in plain collagen sponges and treated with LiCl accumulated significantly more metachromatic matrix than NaCl-treated controls on day 10, and showed a trend towards increased matrix chondroitin-4 sulfate content . CONCLUSIONS: These data suggest that changes in Wnt signaling contribute to chondroinduction of post-natal fibroblasts by DBP . This is the first evidence that Wnt components, which are essential regulators of pre-natal chondrocyte differentiation, may also influence post-natal chondrocyte differentiation induced by DBP.

Toxicol In Vitro, 2004 Aug, 18(4), 533 - 41
Identification of in vitro protein biomarkers of idiosyncratic liver toxicity; Gao J et al.; Drug-induced idiosyncratic hepatotoxicity continues to be an important safety issue for the pharmaceutical industry . This toxicity is due, in part, to the limited predictive nature of current pre-clinical study systems . A hypothesis was formed that treatment of existing in vitro hepatocyte cultures with drugs clinically linked to idiosyncratic hepatotoxicity would result in the release of extracellular protein biomarkers indicative of liver toxicity . To test this hypothesis, a combination of proteomic and immunological techniques were used to first identify, and subsequently verify, components of the protein-laden conditioned culture media from immortalized human hepatocytes which overexpressed cytochrome p450 3A4 . These cells were treated separately with seven individual compounds made up of a combination of thiazolidinedione and l-tyrosine PPARgamma agonists and HIV protease inhibitors, plus a vehicle control (dimethyl sulfoxide) . For each drug class, clinically determined hepatotoxic and non-hepatotoxic compounds were compared . Two proteins, BMS-PTX-265 and BMS-PTX-837, were reproducibly and significantly increased in the conditioned media from cells treated with each of the toxic compounds as compared to media from cells treated with the non-toxic compounds (and vehicle) . This result supported the hypothesis, and so a series of successive assays (western blots and enzyme linked immunosorbent assays) were used to measure the response of these two proteins as a function of an expanded set of 20 compounds . For all 20 drugs, elevations of BMS-PTX-265 correlated exactly with the known safety profile; whereas changes in BMS-PTX-837 correctly predicted the safety profile in 19 of 20 drugs (one false negative) . In summary, the data supports both the pre-clinical in vitro method as a means to identify new biomarkers of liver toxicity, as well as the validity of the biomarkers themselves.

Toxicol In Vitro, 2004 Aug, 18(4), 419 - 25
Cytotoxic effect and role of exogenous antioxidants in carpet dust mediated toxicity in rat hepatocytes in vitro; Ameen M et al.; Carpet industries bear a great deal of economic and commercial significance in India . In order to safe guard the workers against the health hazards caused by dust in their occupational environment; it necessitates studying the biological importance of these dusts . The present study was designed to investigate the toxicity of carpet dust (knotted and tuffted) on isolated rat hepatocytes . The hepatocytes were isolated by collagenase perfusion method and cells were incubated with different concentration of carpet dust (100-5000 microg/10(6) cells) with various time (30-180 min) intervals . An exogenous antioxidant vitamin-E also used to find out the role of antioxidants and free radical production in carpet dust mediated toxicity . Cell viability by trypan blue exclusion and leakage of enzyme lactate dehydrogenase (LDH) were determined . Reduced glutathione (GSH), formation of thiobarbituric acid reactive substance (TBARS) were also measured . A significant decrease in the cell viability was observed after 60, 180 min upon incubation with tuffted carpet dust, while knotted carpet dust caused a significant decrease in the viability after 180 min . LDH leakage was parallel to the cell viability . Thiobarbituric acid reactive substance was significantly increased at 30 and 60 min with carpet dust treated hepatocytes . Dust at 1000 and 5000 microg dose level showed significantly increased formation of TBARS at 30 min incubation . However, when hepatocytes were co-incubated with carpet dust and Vit-E (10, 15 microM), a significant decrease in LDH release and TBARS production was observed while 15 microM Vit-E showed an enhanced protection than 10 microM Vit-E treated hepatocytes . The effect of carpet dust on cell viability, LDH leakage, TBARS production, GSH depletion was time and dose-dependent . Moreover, we observed that tuffted carpet dust causes greater effect than knotted one on the above mentioned parameters . Our studies also revealed that Vit-E in culture media diminishes the carpet dust mediated toxicity.

Zhonghua Jie He He Hu Xi Za Zhi, 2004 Mar, 27(3), 183 - 7
{Study on gene knock-out in Mycobacterium BCG}; Chen XY et al.; OBJECTIVE: To establish the methodology of plasmid for gene knock-out in Mycobacterium BCG . METHODS: We designed two pairs of primers for amplification of MDP1 gene and inserted two fragments into pKO plasmid, and then the recombinant plasmid for MDP1 gene knock-out was obtained, and named pKO-MDP1 . Gene exchange took place within the genome of BCG after pKO-MDP1 plasmid was transformed into Mycobacterium BCG . The strain of Mycobacterium BCG with MDP1 gene knock-out was selected, and the curve of growth rate was studied . RESULTS: The target strain was that of the positive strain by two step PCR and one step sucrose counter selection, without growth in culture media with hygomycin . The value of A(600) per 12 hours was detected for sixteen days . A "S" shaped growth curve was detected . However, there was no significant difference in the growth rates between the wild type Mycobacterium BCG strain and the gene knock-out Mycobacterium BCG strain . CONCLUSIONS: The plasmid of pKO was a useful tool for gene knock-out in Mycobacteria . MDP1 maybe one of the factors influencing the growth rate, but it was not the only one.

Reprod Domest Anim, 2004 Feb, 39(1), 33 - 8
In vitro development of buffalo oocytes in media-containing fluids from different size class follicles; Nandi S et al.; Studies were conducted to investigate the effect of supplementation of fluid from different sized class {small (SFF, < 3 mm), medium (MFF, 3-8 mm) and large (LFF, > 8 mm)} of normal and cystic (CFF) ovarian follicles in oocyte culture media on oocyte maturation rate and embryo development in vitro and to test the efficacy of follicular fluid (FF) from different size classes as a whole oocyte maturation medium . Results suggested that FF were capable of developing buffalo oocytes to embryonic stage in vitro although its efficacy was lower than that of serum . Regardless of high maturation rates after in vitro maturation (IVM) in media containing FF or IVM in whole FF, low blastocyst rates were obtained after in vitro fertilization (IVF) and culture of embryos . Follicular fluid from small follicles had significantly (p < 0.05) higher potential of developing buffalo oocytes to embryonic stage in vitro than that from medium and large follicles . Cystic FF was not capable of supporting development of buffalo oocytes in vitro.

Reprod Domest Anim, 2004 Feb, 39(1), 25 - 32
The influence of opioid peptides on steroidogenesis in porcine granulosa cells; Kaminski T et al.; The present studies were undertaken to examine the influence of mu (beta-endorphin, DAMGO, FK 33-824), delta (met-enkephalin, leu-enkephalin, DPLPE) and kappa opioid receptor agonists (dynorphin A, dynorphin B, U 50488) used at different doses (1-1000 nM) alone and in combination with LH (100 ng/ml) on steroidogenesis in porcine granulosa cells derived from large follicles . The effects of mu, delta and kappa receptor agonists on both basal and LH-induced progesterone (P4) secretion were negligible . Agonists of mu opioid receptors reduced basal androstenedione (A4), testosterone (T) and oestradiol (E2) release . Co-treatment with LH entirely abolished the inhibitory effect of these agonists on A4 and E2 secretion and resulted in an increase in T release . The addition of delta receptor agonists was followed by a decrease in basal A4, T and E2 secretion . The cells incubated in the presence of LH increased the androgen production and abrogated the inhibitory effect of delta agonists on E2 output . Basal A4, T and E2 release was also suppressed by kappa receptor agonists . The presence of LH in culture media extended the inhibitory effect of these opioids on E2 output and caused either abolition of the inhibitory influence of kappa agonists or even augmentation of both androgen release in response to the opioids . In conclusion, these data support the involvement of three major types of opioid receptors in the regulation of porcine granulosa cell steroidogenesis.

J Biomed Mater Res, 2004 Jun 1, 69A(3), 535 - 43
Modulation of differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels modified with Arg-Gly-Asp containing peptides; Shin H et al.; We synthesized biomimetic hydrogels modified with an osteopontin-derived peptide (ODP) and used them as a substrate for in vitro culture of marrow stromal cells (MSCs) to investigate the effect of the biomimetic surface on differentiation of MSCs into osteoblasts . Proliferation and biological assays for 16 days proved that MSCs became differentiated into osteoblasts secreting osteogenic phenotypic markers such as alkaline phosphatase (ALP), osteopontin, and mineralized calcium . In addition, there was an additive effect of the cell-binding peptide on differentiation and mineralization of MSCs cultured in the presence of soluble osteogenic supplements in cell culture media . For example, calcium content at day 16 on peptide-modified hydrogels was significantly higher than on tissue culture polystyrene . Two general trends were observed: (1) proliferation of MSCs decreased as the amount of differentiation markers increased, and (2) higher peptide concentrations accelerated the differentiation of MSCs . On the hydrogel modified with ODP, ALP activity exhibited a maximum value of 36.7 +/- 4.2 pmol/cell/h at day 10 for the concentration of 2 micromol/g while the culture time needed for maximum ALP activity occurred on day 13 for the lower concentrations . On the same hydrogel, the calcium content at day 10 was 21.4 +/- 2.3 ng/cell for the peptide concentration of 2 micromol/g and 1.0 +/- 0.3 ng/cell for 1.0 micromol/g . We used Gly-Arg-Gly-Asp-Ser (GRGDS) for modification of the hydrogel as a comparison to the results with ODP . However, osteoblast development was not significantly affected by the nature of the binding peptide sequences . These results suggest that MSC function can be modulated by variation of the peptide concentration in biomimetic hydrogels used for scaffold-based bone tissue engineering .

J Neurosci Methods, 2004 Jun 15, 136(1), 87 - 98
Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue; Hulse RE et al.; The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function . This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous . Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques . This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 microl . Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha) simultaneously not only from rat serum but, for the first time, also brain tissue . Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

Biomaterials, 2004 Aug, 25(19), 4627 - 32
Inhibition of PMN apoptosis after adherence to dip-coated calcium phosphate surfaces on a NiTi shape memory alloy; Bogdanski D et al.; Nickel-titanium shape-memory alloys (NiTi-SMA) were coated with calcium phosphate by dipping in oversaturated calcium phosphate solution (CaP-coating) . Polymorphonuclear neutrophil granulocytes (PMN) belong to the first cells which will adhere to implant materials . We analyzed the apoptosis of isolated human PMN after cell culture on non-coated and CaP-coated NiTi-SMA by light and scanning electron microscopy (cell morphology) and by flow cytometry (DNA-fragmentation) . In contrast to PMN adherent to non-coated NiTi-SMA, the apoptosis of PMN adherent CaP-coated samples was inhibited . Cell culture media obtained from cultured leukocytes with CaP-coatings (conditioned media, CM) were able to transfer the apoptosis inhibiting activities to freshly isolated PMN . There was a significant {Formula: see text} increase in GM-CSF, IL-8, IL-6, and TNF-alpha within CM obtained from coated versus non-coated NiTi-SMA as was determined by ELISA.

J Med Food, 2004 Spring, 7(1), 114 - 6
Inhibition of Aspergillus parasiticus NRRL 2999 by pollen and propolis extracts; Ozcan M; The inhibitory effects on mycelial growth of Aspergillus parasiticus strain NRRL 2998 of pollen and propolis extracts from different regions of Turkey were investigated in culture media . The least active concentration towards the tested fungus was 2% of both extracts . But, the inhibitory effects of all propolis extracts on mycelial growth were higher when compared with pollen extracts . While the effect of a 5% level of Antakya propolis extract increased with increasing incubation period, the antifungal effect of pollen extract from the same region on mycelial growth was less than that of the control group . As a result, none of the extracts tested completely inhibited mycelial growth . The highest inhibition rate was established at the 5% level of Taskent and Alanya propolis samples.

J Biochem (Tokyo), 2004 Mar, 135(3), 319 - 29
Production of N-lauroylated G protein alpha-subunit in Sf9 insect cells: the type of N-acyl group of Galpha influences G protein-mediated signal transduction; Hashimoto Y et al.; The alpha-subunit of rod photoreceptor G protein transducin (G(t1)alpha) is heterogeneously modified at the N-terminus by a mixture of acyl groups, laurate (C12:0), myristate (C14:0), and two unsaturated fatty acids (C14:1 and C14:2) . Although the N-fatty acylation of G(t1)alpha plays important roles in protein-protein and protein-membrane interactions in light signaling, the biological significance of the heterogeneous acylation remains unclear due to the difficulty in isolating each G(t1)alpha isoform from the retinal rod cells . Here we found that G(t1)alpha/G(i1)alpha chimera (G(t/i)alpha) expressed in Sf9 cells is also heterogeneously modified by myristate (approximately 90%) and laurate (approximately 10%), raising the possibility that the N-acyl group of recombinant G(t/i)alpha may be manipulated by modifying culture media . In fact, addition of myristic acid to the medium decreased the relative content of lauroylated G(t/i)alpha to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated G(t/i)alpha in a concentration-dependent manner . By culturing the G(t/i)alpha-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated G(t/i)alpha, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and approximately 70%, respectively . Functional analysis of these proteins showed that an increase in the relative content of the lauroylated isoform remarkably slowed down the steady-state GTP hydrolysis rate of G(t/i)alpha; the steady-state GTPase activity of the lauroylated isoform was estimated to be one order of magnitude lower than that of the myristoylated isoform . These results suggest that the retinal G(t1)alpha is composed of isoforms having functionally heterogeneous signaling properties.

Ann Thorac Surg, 2004 May, 77(5), 1684 - 9; discussion 1689-90
Myocyte contractility with caspase inhibition and simulated hyperkalemic cardioplegic arrest; Mukherjee R et al.; BACKGROUND: Exposure of left ventricular (LV) myocytes to simulated hyperkalemic cardioplegic arrest (HCA) has been demonstrated to perturb ionic homeostasis and adversely affect myocyte contractility on rewarming . Altered ionic homeostasis can cause cytosolic activation of the caspases . While caspases participate in apoptosis, these proteases can degrade myocyte contractile proteins, and thereby alter myocyte contractility . Accordingly, this study tested the hypothesis that caspase inhibition during HCA would attenuate the degree of myocyte contractile dysfunction upon rewarming, independent of a loss in myocyte viability . METHODS: Porcine (n = 8) LV myocytes were isolated and assigned to the following treatment groups: normothermic control: incubation in cell culture media for 2 hours at 37 degrees C; HCA only: incubation for 2 hours in hypothermic HCA solution (4 degrees C, 24 mEq K(+)); or incubation in hypothermic HCA solution supplemented with 10 microM of the caspase inhibitor, z-VAD (z-Val-Ala-Asp-fluoromethyl-ketone, HCA+zVAD) . Myocyte viability, assayed as a function of mitochondrial function, was determined to be similar in the normothermic and both HCA groups . RESULTS: The HCA caused a significant reduction in myocyte shortening velocity compared with normothermic control values (41 +/- 6 versus 86 +/- 8 microm/s, p < 0.05) . The HCA+zVAD group had significantly improved myocyte shortening velocity compared with the HCA only group (63 +/- 7 microm/s, p < 0.05) . CONCLUSIONS: Independent of changes in viability, caspase inhibition attenuated myocyte contractile dysfunction after HCA and rewarming . Thus, caspase activation during HCA contributes, at least in part, to impaired myocyte contractility with rewarming . Supplementation of HCA with caspase inhibitors may provide a means to preserve myocyte contractile function after cardioplegic arrest.

Transplant Proc, 2004 Apr, 36(3), 695 - 7
Effect of calcineurin inhibitors on extracellular matrix turnover in isolated human glomeruli; Esposito C et al.; INTRODUCTION: Although chronic cyclosporine toxicity is mainly characterized by tubular atrophy and interstitial fibrosis, glomerular injury with expansion of mesangial matrix and sclerosis is not uncommon . Tacrolimus is a newer calcineurin inhibitor that has been used in renal transplant recipients as primary or rescue therapy . Clinical trials suggest an improved long-term graft survival among patients treated with tacrolimus . Recently we have shown that tacrolimus and cyclosporine have similar effects on extracellular matrix turnover in cultured cells . The present study was performed to investigate the effects of the calcineurin inhibitors on whole glomeruli extracellular matrix turnover . METHODS: Human glomeruli isolated from kidney biopsies just before transplantation were incubated with culture media containing either cyclosporine (200 ng/mL) or tacrolimus (10 ng/mL) for 24 hours . Glomeruli incubated only with culture medium were used as control . RESULTS: The expressions of (alpha2)IV collagen, metalloprotease 9 (MMP9), tissue inhibitors of metalloproteases 2 (TIMP-2), and TGFbeta were evaluated by in situ reverse transcription and polymerase chain reactions (RT-PCR) . beta-actin was used as a control gene . Cyclosporine (but not tacrolimus) increased the expression of (alpha2)IV collagen and TIMP2 in isolated glomeruli . TGF-beta was markedly increased by cyclosporine . MMP9 expression was not affected by the calcineurin inhibitors . By light microscopy kidney biopsies did not show pathologic changes . CONCLUSION: Cyclosporine treatment modulates extracellular matrix turnover in isolated human glomeruli, inducing an imbalance between synthesis and degradation . This effect, not observed in tacrolimus-treated human glomeruli, may induce the extracellular matrix deposition and sclerosis characteristic of chronic cyclosporine toxicity.

Transplant Proc, 2004 Apr, 36(3), 607 - 8
In vitro modulation of monocyte chemoattractant protein-1 release in human pancreatic islets; Marzorati S et al.; Islet transplantation is a new approach to treat type 1 diabetic patients . Despite its great potential and progressively increasing success rate, islet engraftment still represents an unsolved problem . Only part of the transplanted beta-cell mass survives after infusion due to hypoxia and inflammatory reactions, principally mediated by macrophages . We have demonstrated that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of a transplant . In this study we have attempted to modulate in vitro MCP-1 release by human islets . Human islets isolated using the automated method were cultured in CMRL or M199 standard culture media alone or supplemented with (1) two intracellular kinase inhibitors (10 micromol/L RO8220, a protein kinase C inhibitor, and rcAMP 20 micromol/L, a protein kinase A inhibitor) or (2) two antioxidant and cell-protective agents (vitamin E, vitamin B); or (3) immunosuppressive drugs (0.001 to 10 ng/mL cyclosporine, 0.1 to 100 ng/mL rapamycin, 0.1 to 10 ng/mL tacrolimus, 0.001 to 10 ng/mL mycophenolate acid) . We observed that the only culture condition that significantly decreased MCP-1 in human islets were CMRL (31 +/- 12 in CMRL vs 539 +/- 184 pg/mL, in M199, P <.05) or cyclosporine (514 +/- 83 pg/mL in control islet vs 307 +/- 13, 231 +/- 44, 192 +/- 4, 242 +/- 113, 169 +/- 15 pg/mL in islet plus cyclosporine ranging from 0.001 to 10 ng/mL, respectively, P >.05) . The capacity of in vitro factors to decrease human islet MCP-1 release suggests strategies to increase the success of islet transplantation.

Aquat Toxicol, 2004 May 28, 68(1), 61 - 74
Long-term acclimation of Pseudokirchneriella subcapitata (Korshikov) Hindak to different copper concentrations: changes in tolerance and physiology; Bossuyt BT et al.; The effect of long-term copper acclimation of the freshwater green algae Pseudokirchneriella subcapitata to copper was investigated using different physiological and toxicological endpoints . The algae were exposed to seven-five of which are ecologically relevant for European surface waters-copper concentration ranging from 0.5 to 100 microgCul(-1) during a 3-month period . A standard medium was used as culture and test medium with an addition of 2 mg DOCl(-1) (replacing EDTA) . At certain intervals, experiments were performed to assess algal biomass, growth rate, chlorophyll and carotenoid content, pigment diversity, autotrophic index, intracellular and adsorbed copper, and the sensitivity of the algae to copper . Chronic copper tolerance (mean +/- standard deviation) increased significantly from 88 +/- 15 to 124 +/- 25 microg Cul(-1) for P . subcapitata acclimated to 0.5 and 100 microg Cul(-1), respectively . Based on the algal biomass, the growth rate, the pigment diversity and the autotrophic index, an optimal concentration range was observed between 1 and 35 microg Cul(-1) . Significant decreases in algal biomass, pigment diversity and autotrophic index were observed in algal cultures acclimated to 0.5 microg Cul(-1) and 100 microg Cul(-1) . Chlorophyll a content (mean +/- standard deviation) increased from 8.4 +/- 3.1 to 28.6 +/- 7.5 x 10(-14) g per cell and carotenoid content (mean +/- standard deviation) increased from 3.7 +/- 0.8 to 7.1 +/- 1.2 x 10(-14) g per cell for algae exposed to 1 and 100 microg Cul(-1), respectively . Intracellular copper increased from 0.099 to 20.6 x 10(-15) g Cu per cell and adsorbed copper increased from 0.026 to 1.8 x 10(-15) g Cu per cell for algae acclimated for 12 weeks to 0.5 and 100 microg Cul(-1), respectively . This research demonstrates that the use of standard culture media, some of which may be deficient in copper, can result in sub-optimal performance of the organisms, which in turn may affect toxicity test results . Additionally, this work also established an optimal concentration range for copper for this algal species . This phenomenon should be taken in consideration when performing environmental risk assessments of essential elements .

Cloning Stem Cells, 2004, 6(1), 15 - 23
M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo; Bogliolo L et al.; This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo . Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h . In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment . Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF . After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media . MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo . This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases . Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.

Cochrane Database Syst Rev . 2004;(2):CD004378.
Day three versus day two embryo transfer following in vitro fertilization or intracytoplasmic sperm injection; Oatway C et al.; BACKGROUND: Embryo transfer (ET) was traditionally performed two days after oocyte retrieval; however, developments in culture media have allowed embryos to be maintained in culture for longer periods . Delaying transfer from day two to day three would allow for further development of the embryo and might have a positive effect on pregnancy outcomes . OBJECTIVES: To determine if there is any difference in live birth and pregnancy rates when ET is performed on day three, compared with day two, in infertile couples undergoing treatment with in vitro fertilization (IVF), including intracytoplasmic sperm injection (ICSI) . SEARCH STRATEGY: We searched the Cochrane Menstrual Disorders & Subfertility Group trials register (17th December 2003), the Cochrane Central Register of Controlled Trials (The Cochrane Library Issue 1, 2003), MEDLINE (1966 to 2003), EMBASE (1980 to 2003) and Biological Abstracts Databases (1980 to 2003), the National Research Register (NRR), the Medical Research Council's Clinical Trials Register, the NHS Centre for Reviews and Dissemination, citation lists of relevant publications, review articles and included studies, as well as abstracts of appropriate scientific meetings . SELECTION CRITERIA: Randomized controlled trials that compared day three versus day two embryo transfer after oocyte retrieval during an IVF or ICSI treatment cycle in subfertile couples . DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed trial quality and extracted data . Study authors were contacted for additional information . The primary outcome measures were live birth rate and ongoing pregnancy rate . MAIN RESULTS: Ten studies involving 2027 women were included, but only three studies reported live birth and four reported ongoing pregnancy rates . The pooled odds ratios (day three compared to day two) were 1.07, 95% CI 0.84 to 1.37 for live birth and 1.05, 95% CI 0.83 to 1.32 for ongoing pregnancy . From ten studies, the pooled odds ratio for clinical pregnancy was 1.26, 95% CI 1.06 to 1.51 . Sub-group analysis revealed that this advantage occurred in those undergoing ICSI cycles . A higher miscarriage rate with day 3 ET in ICSI cycles works to negate the increased clinical pregnancy rate, in agreement with the finding of no significant difference between treatments in live birth rate . REVIEWERS' CONCLUSIONS: Although an increase in clinical pregnancy rate with day three embryo transfer was demonstrated, at present there is not sufficient good quality evidence to suggest an improvement in live birth when embryo transfer is delayed from day two to day three.

Life Sci, 2004 May 21, 75(1), 21 - 34
Production of prostaglandinE2 via bile acid is enhanced by trypsin and acid in normal human esophageal epithelial cells; Kawabe A et al.; Several reports suggest that duodenogastroesophageal reflux may produce esophagitis, Barrett's esophagus and esophageal carcinoma . And it is well known that the incidence of adenocarcinoma arising from Barrett's esophagus has been increasing during the past decade . On the other hand, cyclooxygenase-2 and prostaglandins, produced by the catalytic reaction of cyclooxygenase-2, are considered to relate to carcinogenesis of the digestive tract and other malignant tumors . Recent reports suggest that cyclooxygenase-2 is induced in Barrett's esophagus and esophageal carcinoma . The purpose of this study is to investigate the reaction of cyclooxygenase-2 expression and prostaglandinE2 production on normal human esophageal epithelial cells cultured with gastroduodenal components . Normal human esophageal epithelial cells were cultured with chenodeoxycholic acid, trypsin and in acidic condition, individually and with different combinations of these three factors . After culturing, cyclooxygenase-2 expression in the cells and amount of prostglandinE2 in culture media was evaluated by immunoblotting and enzyme-immunoassay, respectively after culturing the cells . Cyclooxygenase-2 expression was up-regulated by bile acid and prostaglandinE2 production was enhanced by bile acid with trypsin, acidic condition or both of these components, without a synergistic effect on cyclooxygenase-2 expression . Production of prostaglandinE2 via these factors was suppressed by the cyclooxygenase-2 selective inhibitor JTE-522.The results suggest that duodenogastroesophageal reflux may induce cyclooxygenase-2 expression and prostaglandinE2 production in esophageal epithelial cells, cyclooxygenase-2 specific inhibitors may have a chemopreventive effect on esophageal carcinoma.

Environ Pollut, 1989, 57(2), 103 - 15
Distribution of planktonic bacteria capable of degrading sodium dodecyl sulphate (SDS) in a polluted South Wales river; White GF et al.; The temporal and geographical distributions of planktonic bacteria, and their ability to degrade sodium dodecyl sulphate (SDS) (AS(+) phenotype), in a polluted South Wales river, is reported for five sites source to estuary sampled during a one-year period . The annual mean prevalence of AS(+) isolates at all sites was 8.1-16.0% of the total number of isolates, and these values were not altered by including SDS in culture media . Although the proportion of AS(+) isolates in clean water at the source was not significantly different to that of polluted sites, the AS(+) cell density was lower at the source because of its lower overall numbers . The percentage of AS(+) isolates in estuarine water was higher than at the three polluted mid-river sites, but when cell numbers were taken into account, the AS(+) cell density was the same at all polluted sites including the estuary . There was no correlation between percentage of AS(+) and either BOD or oxygen concentration, but AS(+) isolates were significantly more prevalent at the end of summer . Of the AS(+) isolates, more than half contained constitute alkysulphate enzymes, the remainder being induced or repressed by SDS; the relative proportions of enzyme regulatory type did not vary significant between sites or at different sampling times.

Environ Pollut, 1991, 74(2), 89 - 100
The influence of pH on cadmium toxicity to the green alga Stichococcus bacillaris and on the cadmium forms present in the culture medium; Skowronski T et al.; Cadmium toxicity to the green alga Stichococcus bacillaris was investigated in media of pH 3-9 . A significant decrease of cadmium toxicity occurred in both the acidic and alkaline ranges of pH . In media of pH 3 and 9, cadmium did not affect the dry mass content substantially . Maximum toxicity of cadmium was noticed at pH 6-7 . Voltametric investigations showed a significant effect of pH on electrochemically measured cadmium content in the culture media . Hydrolysis of the medium components and formation of cadmium complexes with OH(-) ions caused a considerable decrease in amounts of electrochemically measured cadmium in the alkaline range of pH.

Eur J Med Res, 2004 Feb 27, 9(2), 71 - 7
Effect of extracellular hypertonicity and alkalosis on endothelial-derived EA.hy 926 cells in vitro; Hirsch C et al.; Endothelial and local metabolic mechanisms contribute in concert to the regulation of blood flow . In vivo extracellular alkalosis induces a vasoconstriction, hyperosmolarity a vasodilatation . The interaction between local metabolic and endothelial mechanisms is poorly understood . Therefore we investigated in endothelial-derived EA.hy926 cells the secretion of endothelial modulators of vascular tone under hypertonic stress with and without alkalosis: hyperosmolality was generated by either the addition of NaHCO subset 3 (25, 50, 100 mM, pH up to > 8) or mannitol (50, 100, 200 mM) to the cell culture media . The cells were studied using automated cell counting, measurement of the activity of the lactate dehydrogenase (LDH) and a bromo-deoxyuridine (BrdU) cell proliferation assay . Endothelin and cGMP, a surrogate marker for nitric oxide (NO), were measured with specific ELISAs . EA.hy 926 cells formed stable monolayers in vitro . The secretion of endothelin, but not of cGMP was inversely correlated with the osmolality of the incubation media: the endothelin concentration in the supernatants decreased in both mannitol- and NaHCO subset 3 -treated cells in a concentration-dependent manner (152.4 +/- 6.2 pg/ml (control) to 24.4 +/- 2.4 pg/ml (200 mM mannitol), res . to 18.2 +/- 2.7 pg/ml (100 mM NaHCO subset 3) . Neither hypertonic bicarbonate nor mannitol solutions decreased the monolayer cell density or cell viability during the 6 hour incubation period . In conclusion, EA.hy926 cells are quite resistant against a 6-hour hypertonic/alkaline stress . Hypertonicity decreases the secretion of endothelin and has no effect on cGMP . At each level of hypertonicity the endothelin concentration was similar in the NaHCO subset 3 and mannitol media arguing against a direct role of endothelin in alkalosis-induced vasoconstriction in vivo . The decreased secretion of endothelin during hypertonicity could contribute to the hyperosmolal vasodilation seen in vivo.

What Is Bioreactor?, What Is Activated Sludge?, What Is Listeria Monocytogenes?, What Is Rhizobia?, What Is Amino Acid?, c, Bacterium, a, Bacteria, i, Microorganisms, e, Microbiology, o, Microbes, a, Escherichia coli, e, Campylobacter, c, Yeasts, c, Rhizobacterium, c, Antimicrobials, o, Microorganisms, c, Acinetobacter, r, Cryptococci, n, Corynebacter, e, Enterobacters, i, Wastewater, c, Escherichia coli, r, Antibiotics, i, Cryptococci, i, Escherichia coli, n, Antibiotic prophylaxis, s, Bioremediation, r, Microbial, n, Escherichia coli, r, Microorganism, r, Acinetobacter

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005