J Cereb Blood Flow Metab, 2004 Apr, 24(4), 409 - 18 Effects of interleukin-1beta and prostaglandin E2 on prostaglandin D synthase production in cultivated rat leptomeningeal cells; Muraki T et al.; Although the interleukin (IL)-1 receptor is densely distributed in the leptomeninges constituting the blood/cerebrospinal fluid barrier, its physiologic significance has remained unclear . In the present study, we show that in cultured leptomeningeal cells, IL-1beta, tumor necrosis factors, or lipopolysaccharide causes a prominent increase in the synthesis and release of prostaglandin (PG) D synthase, which catalyzes the final step in the biosynthesis of PGD2 . Although significant increases in the amount of PGD synthase were also observed with cells exposed to somatostatin, thrombin, or ciliary neurotrophic factor, these were much smaller than were those induced by the proinflammatory cytokines . Other agents tested including IGF-I had no effect upon the enzyme levels in the culture media . Furthermore, we found that the increased secretion of PGD synthase by IL-1beta was completely inhibited by 10(-7) M PGE2 . The same dose of PGD2 or 15-deoxy-Delta(12-14)PGJ2 had no effect upon the IL-1beta action . In addition, PGE2 increased the level of fibronectin and eliminated the expression of zonula occludentes-1, a tight junction-associated protein from cultured cells, effects likely reflecting a loss of barrier integrity . These results demonstrate the importance of inflammatory stimuli as a physiologic regulator of the leptomeningeal cell function. J Neurochem, 2004 May, 89(3), 674 - 84 Comparison of in vivo and in vitro subcellular localization of estrogen receptors alpha and beta in oligodendrocytes; Zhang Z et al.; The existence of estrogen receptors (ERs) in oligodendrocytes (OLGs) in vivo and in vitro is unresolved, as their presence has been reported in some studies and their absence in others . Using molecular and immunocytochemical techniques, we describe the subcellular localization of ERalpha and ERbeta in OLGs in vivo and in vitro . Both ERalpha and ERbeta are detected in an immortalized OLG cell line and in enriched OLG cultures by RT-PCR and western blot . Immunocytochemistry of OLGs from enriched cultures shows ERalpha receptors are nuclear, whereas ERbeta receptors are cytoplasmic . Confocal and deconvolution microscopy of enriched OLG cultures reveals ERbeta immunoreactivity is concentrated in perikarya and veins of OLG membrane sheets; lesser reactivity is present in their plasma membranes and nuclei . In vivo, we readily detect ERalpha in neurons but not in OLGs, even though we used different fixation procedures and different ERalpha antibodies . The presence of ERalpha in cultured OLGs may be due to culture media that contains factors stimulating ERalpha expression but are reduced in normal brain . In vivo, ERbeta immunoreactivity is readily detectable in OLG cytoplasm and in myelin sheaths . Incubation of glial cultures without or with increasing concentrations of 17beta-estradiol (E2) shows that E2 significantly accelerates OLG process formation. Biochem Pharmacol, 2004 May 1, 67(9), 1677 - 88 Role of zinc and iron chelation in apoptosis mediated by tachpyridine, an anti-cancer iron chelator; Zhao R et al.; Tachpyridine (N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane; tachpyr) is a potent hexadentate iron chelator under preclinical investigation as a potential anti-cancer agent . Tachpyridine induces apoptosis in cultured cancer cells by triggering a mitochondrial pathway of cell death that is p53-independent . To explore the relationship between the chelation chemistry of tachpyridine and its biological activity, a sensitive and specific reversed-phase high-performance liquid chromatography (RP-HPLC) method was devised and used to measure tachpyr and its metal complexes in cells and tissue culture media . Major species identified in cells treated with tachpyr were tachpyr itself, {Zn(tachpyr)}(2+), and iron coordinated to two partially oxidized species of tachpyridine, {Fe(tachpyr-ox-2)}(2+), and {Fe(tachpyr-ox-4)}(2+) . The kinetics of intracellular accumulation of {Zn(tachpyr)}(2+) and {Fe(tachpyr-ox-2)}(2+) were markedly different: {Zn(tachpyr)}(2+) rapidly reached plateau levels, whereas intracellular levels of {Fe(tachpyr-ox-2)}(2+) and free tachpyr rose steadily . At the last timepoint measured, 9% of total cellular iron and 13% of total cellular zinc were bound by tachpyridine . Taken together, {Zn(tachpyr)}(2+), {Fe(tachpyr-ox-2)}(2+), and free tachpyr accounted for virtually all of the tachpyr added, indicating that iron and zinc are the principal metals targeted by tachpyridine in cells . Consistent with these findings, activation of the apoptotic caspases 9 and 3 was blocked in cells pre-treated with either iron or zinc . Pretreatment with either of these metals also completely protected cells from the cytotoxic effects of tachpyridine . These results demonstrate a link between metal depletion and chelator cytotoxicity, and suggest that intracellular chelation of zinc as well as iron may play a role in the cytotoxicity of tachpyridine. J Control Release, 2004 Apr 28, 96(2), 325 - 40 In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers; Turk M et al.; In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media . First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa) . Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature . Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers . A green fluorescent protein expressing plasmid was used as a model . Complexes of this plasmid both with PEIs and their copolymers were formed . The zeta potentials of these complexes were between -3.1 and +21.3 . Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs . Copolymerization caused a profound decrease in the positive charges . Particle sizes of the complexes were in the range of 190-1235 nm . Using high polymer/plasmid ratios caused aggregation . The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight . Copolymers were able to squeeze plasmid DNA more at the body temperature . Cytotoxicity was observed with PEIs especially with the branched higher molecular weights . Copolymerization reduced the cytotoxicity . The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PEI25B . However, poly(NIPA)/PEI25L was the most successful vector for an effective gene expression without any significant toxicity. BMC Cell Biol . 2004 Apr 06;5(1):14. Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function; Malina HZ et al.; BACKGROUND: Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria . Its accumulation can be observed in aging-related diseases, e.g . senile cataract and infectious disease . We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid . RESULTS: Xanthurenic acid at 10 or 20 microM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria . In 20 microM xanthurenic acid, Bax is also translocated to the nucleus . In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption . CONCLUSION: Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions. Anal Bioanal Chem, 1996 Mar, 354(5-6), 624 - 8 Soil bacteria sensitivity towards heavy metals - Experimental system optimisation using chemical speciation calculations; Geiger G et al.; The ecotoxicological relevance of many laboratory studies on soil bacteria sensitivity towards heavy metals is limited because culture conditions are chosen which do not adequately represent field conditions . The influence of the composition of culture media on the speciation of copper in an oligotrophic model soil system has been investigated . The expected chemical speciation has been calculated to obtain information on the concentration of the bioavailable fraction and the totally dissolved copper . For control measurements of totally dissolved copper, vacuum filtration has been used . The results of the measurements are in a good agreement with the calculations . Therefore, the use of speciation calculations is postulated as a useful tool for the assessment of free metal concentrations at higher pH, where the concentrations of dissolved metals are too low to be measured by simple methods. Plant Physiol Biochem, 2004 Jan, 42(1), 35 - 42 Spatial expression of a sunflower SERK gene during induction of somatic embryogenesis and shoot organogenesis; Thomas C et al.; Organogenesis or somatic embryogenesis can be induced on immature zygotic embryos (IZE) of sunflower depending on the culture conditions . Both morphogenic processes originate from the same group of cells and show identical kinetics . Using real-time PCR and in situ hybridisation, we showed that somatic embryogenesis receptor-like kinase (SERK) transcripts accumulate early after the beginning of the culture in the morphogenic zone of IZE explants whatever the induction conditions used, i.e . organogenic, embryogenic or highly embryogenic conditions . Quantitative analyses failed to show any correlation between the SERK expression level during the period decisive for the orientation of the morphogenic pathway, i.e . the first 2 days of culture, and the type of morphogenesis induced . However, after 2 days of culture on the organogenic medium, the SERK gene expression level was severely down-regulated in the IZE explants . At 4 days of culture, SERK transcripts were no longer detectable by in situ hybridisation in the developing shoot structures whereas they still continued to accumulate in the embryonic structures induced on both embryogenic and highly embryogenic culture media . The significance of these expression analyses was addressed by transfer medium experiments . Results revealed that IZE cultured on the organogenic medium were able to form somatic embryos when transferred on the highly embryogenic medium as long as the SERK transcripts accumulated at a high level in their morphogenic zone, i.e . first 2 days of culture . Passt this delay, explants rapidly lost their embryogenic competence . Indeed, after 4 days of culture on the organogenic medium, IZE were definitely oriented towards shoot organogenesis . Taken together, these data suggest that reactive cells of IZE develop the competence to somatic embryogenesis during the first day of culture whatever the morphogenic induction conditions used. Cancer Res, 2004 Apr 1, 64(7), 2397 - 405 Gene expression profile of gastric carcinoma: identification of genes and tags potentially involved in invasion, metastasis, and carcinogenesis by serial analysis of gene expression; Oue N et al.; Gastric carcinoma (GC) is one of the most common malignancies worldwide . To better understand the genetic basis of this disease, we performed serial analysis of gene expression (SAGE) on four primary GC samples and one associated lymph node metastasis . We obtained a total of 137,706 expressed tags (Gene Expression Omnibus accession number GSE 545, SAGE Hiroshima gastric cancer tissue), including 38,903 that were unique . Comparing tags from our GC libraries containing different stages and different histologies, we found several genes and tags that are potentially involved in invasion, metastasis, and carcinogenesis . Among these, we selected 27 genes and measured mRNA expression levels in an additional 46 GC samples by quantitative reverse transcription-PCR . Frequently overexpressed genes (tumor/normal ratio > 2) were COL1A1 (percentage of cases with overexpression, 78.3%), CDH17 (73.9%), APOC1 (67.4%), COL1A2 (58.7%), YF13H12 (52.2%), CEACAM6 (50.0%), APOE (50.0%), REGIV (47.8%), S100A11 (41.3%), and FUS (41.3%) . Among these genes, mRNA expression levels of CDH17 and APOE were associated with depth of tumor invasion (P = 0.0060 and P = 0.0139, respectively), and those of FUS and APOE were associated with degree of lymph node metastasis (P = 0.0416 and P = 0.0006, respectively) . In addition, mRNA expression levels of FUS, COL1A1, COL1A2, and APOE were associated with stage (P = 0.0414, P = 0.0156, P = 0.0395, and P = 0.0125, respectively) . Quantitative reverse transcription-PCR analysis also showed a high level of REGIV expression (>100 arbitrary units) in 14 of 46 GC samples (30.4%) but not in noncancerous tissues . We detected V5-tagged RegIV protein in the culture media of cells transfected with pcDNA-RegIV-V5 by Western blot . Our results provide a list of candidate genes that are potentially involved in invasion, metastasis, and carcinogenesis of GC . REGIV may serve as a specific biomarker for GC. Am J Ophthalmol, 2004 Apr, 137(4), 662 - 7 The challenge of determining aqueous contamination rate in anterior segment intraocular surgery; Ta CN et al.; PURPOSE: To determine aqueous contamination rate in anterior segment intraocular surgery using two different techniques of obtaining aqueous fluid and to assess whether a 3-day application of topical 0.3% ofloxacin reduces this contamination rate compared with a 1-hour application . DESIGN: Randomized clinical trial . METHODS: One hundred and thirty-three eyes of 130 patients undergoing anterior segment intraocular surgery were randomized to either control (64 eyes received topical ofloxacin 1 hour before surgery) or study groups (69 eyes received topical ofloxacin four times a day for 3 days before surgery in addition to 1 hour preoperatively) . Eyes in both groups received a periorbital iodine scrub and two drops of topical 5% iodine . Aqueous fluid was obtained at the beginning and conclusion of surgery using a cannula passed through a paracentesis or a needle passed through clear cornea . The aqueous, cannula, and needles were inoculated in blood culture media broth and bacterial growth was identified . RESULTS: Overall, eight of 89 aqueous samples (9%) obtained using a cannula at the beginning of surgery were culture-positive . Similarly, six of 41 aqueous samples (15%) obtained through a needle through clear cornea at the beginning of surgery showed contamination . At the conclusion of surgery, nine of 112 samples (8%) showed positive cultures . There was no difference in the aqueous contamination rates between the control and study groups . CONCLUSIONS: Despite the use of a needle to obtain aqueous fluid at the beginning of surgery before creating a paracentesis, the aqueous contamination rate remained higher than that found at the conclusion of surgery . A 3-day application of topical ofloxacin before surgery did not reduce the anterior chamber aqueous contamination rate relative to a 1-hour application. Reproduction, 2004 Feb, 127(2), 187 - 94 Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection; Choi YH et al.; This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI) . The effect of manipulation temperature (room temperature vs 37 degrees C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated . The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61-72 vs 23-25% respectively) . Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57-59% respectively) . For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55-61% respectively) . The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes . Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI . Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05) . Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes . These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media. J Neurochem, 2004 Apr, 89(2), 464 - 73 Astrocytic production of nerve growth factor in motor neuron apoptosis: implications for amyotrophic lateral sclerosis; Pehar M et al.; Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS) . We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine . Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium . Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer . Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies . In addition, nitric oxide synthase inhibitors were also protective . Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide . NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos . Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide . This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms . Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation . Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS. Ned Tijdschr Geneeskd, 2004 Mar 13, 148(11), 533 - 6 {Cutaneous nocardiosis as an opportunistic infection}; Bogaard HJ et al.; A 46-year-old man who had been treated with azathioprine and budesonide for Crohn's disease for the past eight years developed a purulent skin condition on the right ring finger . Despite surgical drainage and treatment with amoxicillin and flucloxacillin, the condition spread itself over the hand and lower arm, partly per continuum and partly in jumps . The patient did not feel ill and there were no systemic symptoms . Ultimately, Nocardia asteroides was cultured from the wound and complete cure was achieved after 8 months' treatment with co-trimoxazole . Infections with Nocardia spp . are rare but may occur more often and run a more fulminant course in patients under treatment with immunosuppressants . Cutaneous nocardiosis generally has a characteristic lymphogenous spreading pattern, but an atypical picture with pustules, pyoderma, cellulitis or abscess formation is also possible . In non-cutaneous nocardiosis there is usually pneumonia or lung abscess, possibly with secondary haematogenous spread to the central nervous system or skin . Culturing Nocardia requires more time than usual but can be promoted by special culture media . Treatment of the infection with co-trimoxazole is the method of choice and is almost always successful in cases of cutaneous nocardiosis. Eur J Gynaecol Oncol, 2004, 25(1), 33 - 9 Effect of cis-diammine dichloroplatinum on vascular endothelial growth factor expression in uterine cervical carcinoma; Miyahara Y et al.; PURPOSE OF INVESTIGATION: In this study, we investigated the effects of cis-diammine dichloroplatinum (CDDP) on VEGF mRNA expression and VEGF production in uterine cervical carcinoma tissues obtained from patients with locally advanced disease and in CaSki cells cultured in vitro . METHODS: VEGF in cultured CaSki cells and in the culture media was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) before and 24 h, 48 h and 72 h after 3 h exposure to CDDP . VEGF mRNA expression in CaSki cells was assessed by the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and 24 h and 48 h after 3 h exposure to CDDP . We also examined the effect of CDDP on microvessel counts in uterine cervical carcinoma tissues obtained before and after high-dose CDDP intraarterial chemotherapy . Immunohistochemical staining using a monoclonal antibody against CD34 was carried out with cervical carcinoma tissue specimens, and microvessel counts were quantified by counting vessels . RESULTS: CDDP treatment resulted in significant increases in not only VEGF concentrations in cultured CaSki cells and culture media but also in VEGF mRNA expression levels in cultured CaSki cells in a time-dependent and dose-dependent manner compared to untreated controls (p < 0.05, n = 5) . On the other hand, VEGF concentrations and microvessel counts in cervical carcinoma tissues were significantly lower in cases with complete response (CR) and partial response (PR), compared to those before treatment (p < 0.05, n = 5 ) . By contrast, in cases with no change (NC) to CDDP, both VEGF concentrations and microvessel counts did not decrease and rather showed a somewhat increase compared with levels prior to the treatment . CONCLUSIONS: These results suggest that CDDP-induced increases in VEGF production by cervical carcinoma cells may stimulate angiogenesis in the tumor lesion after CDDP treatment. Biotechnol Appl Biochem, 2005 Feb, 41(Pt 1), 67 - 76 Influence of culture conditions on the N-glycosylation of a monoclonal antibody specific for recombinant hepatitis B surface antigen; Cabrera G et al.; MAbs (monoclonal antibodies) are becoming increasingly important as diagnostic tools for pharmaceutical biotechnology, and hence it is crucial that they are produced under controlled conditions to assure their consistency and reproducibility, not only in terms of protein sequence and bioactivity, but also in terms of post-translational modifications, e.g . for N-glycosylation . Hybridoma CB.Hep-1, which secretes an IgG2b mAb, was cultured in vivo in ascites and in vitro in static-flask, spinner-flask, dialysis-membrane and perfusion systems using protein-free, low-serum-containing medium (1% foetal-calf serum) and high-serum-containing medium (8% foetal-calf serum) . These CB.Hep-1 mAbs were fully characterized, and insignificant differences in the affinity constant were observed . Glycosylation profiling was performed by labelling the N-glycans released by peptide N-glycosidase F with either of the fluorophore tags 8-aminonaphthalene-1,3,6-trisulphonic acid and 4-aminobenzoic acid . The mAb produced in vivo showed two major biantennary-complex-type N-glycans: monogalactosylated, core-fucosylated and agalactosylated, core-fucosylated . The mAbs produced in vitro in static flasks and spinner flasks were not significantly influenced by the serum content in the culture media and showed a higher degree of N-glycan galactosylation compared with those produced in mouse-ascites, hollow-fibre and membrane systems . The monogalactosylated, core-fucosylated structure was the most abundant N-glycan except for those produced in ascites and hollow fibres, where the agalactosylated, core-fucosylated glycoform was the major specie . MAbs produced in high-cellular-yield systems displayed greater galactosylation heterogeneity influenced by changes in culture media. Pol J Pharmacol, 2004 Jan-Feb, 56(1), 129 - 36 Immunophilin ligands decrease release of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-2 in rat astrocyte cultures exposed to simulated ischemia in vitro; Gabryel B et al.; The aim of present study was to evaluate the effects of immunophilin ligands (cyclosporin A, FK506 and rapamycin) on the simulated ischemia-induced release of pro-inflammatory cytokines (IL-1beta, TNF-alpha and IL-2) in rat primary astrocyte cell cultures . Astrocytes were exposed to cyclosporin A (CsA) (0.25, 0.5, 1, 10, 20 and 50 microM), FK506 (1, 10, 100, 1000 nM) and rapamycin (10, 100, 500 and 1000 nM) . In vitro simulated ischemia significantly increased secretion of IL-1beta, TNF-alpha and IL-2 by astrocyte cultures deprived of microglia (by shaking and incubating with L-leucine methyl ester) . CsA (at concentrations of 10-50 microM), FK506 (at all used concentrations) and rapamycin (in dose-dependent manner) significantly attenuated IL-1beta release after 24 h exposure to ischemic conditions . Immunophilin ligands at all used concentrations significantly decreased TNF-alpha levels in culture media after 24 h exposure to ischemia . Moreover, significant decrease in IL-2 secretion at 0.25, 0.5, 1 and 50 microM CsA and FK506 at concentrations of 100 and 1000 nM were observed . The results suggest that immunophilin ligands may regulate glial activity during ischemia by affecting the release of pro-inflammatory cytokines. Plant Cell Physiol, 2004 Mar, 45(3), 265 - 74 3-methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions; Takatsuka C et al.; Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation . When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells . Therefore, using light microscopy, we can determine whether cells have performed autophagy . In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells . The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c . We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy . 3-MA blocked cellular protein degradation without any effect on cellular protease activity . In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA . The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells . These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells. Biomaterials, 2004 Aug, 25(18), 4175 - 83 Increased viable osteoblast density in the presence of nanophase compared to conventional alumina and titania particles; Gutwein LG et al.; In the present in vitro study, osteoblast (bone-forming cells) viability and cell density were investigated when cultured in the presence of nanophase compared to conventional (i.e . micron) alumina and titania particles at various concentrations (from 10,000 to 100 microg/ml of cell culture media) for up to 6h . Results confirmed previous studies of the detrimental influences of all ceramic particulates on osteoblast viability and cell densities . For the first time, however, results provided evidence of increased apoptotic cell death when cultured in the presence of conventional compared to nanophase alumina and titania particles . Moreover, since material characterization studies revealed that the only difference between respective ceramic particles was nanometer- and conventional-dimensions (specifically, phase and chemical properties were similar between respective nanophase and conventional alumina as well as titania particles), these results indicated that osteoblast viability and densities were influenced solely by particle size . Such nanometer particulate wear debris may result from friction between articulating components of orthopedic implants composed of novel nanophase ceramic materials . Results of a less detrimental effect of nanometer--as compared to conventional-dimensioned particles on the functions of osteoblasts provide additional evidence that nanophase ceramics may become the next generation of bone prosthetic materials with increased efficacy and, thus, deserve further testing. Biochem Biophys Res Commun, 2004 Apr 16, 316(4), 991 - 6 High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells; Lim LH et al.; Blo t 5 is a major allergen from house dust mite Blomia tropicalis . Purification of native Blo t 5 (nBlo t 5) from whole dust mite extract is tedious and gave low yield . In this study, we demonstrated that codon optimization facilitated high-level expression of Blo t 5 in Chinese hamster ovary (CHO)-K1 cells and thus allows production of sufficient recombinant cBlo t 5 for specific immunotherapy . A codon optimized Blo t 5 gene was synthesized by PCR and the codon optimized or wild-type Blo t 5 gene in pcDNA3.0 was transfected into CHO-K1 cells and stably selected with Geneticin (G418) . Western-immunoblot analysis of spent culture media detected a positive band at 14kDa for the codon optimized but not wild-type gene transfectants . In addition, a stable CHO-K1 clone produced up to 13 mg/L of the cBlo t 5 protein having a high correlation of human IgE reactivities and allergenicity to the native Blo t 5, thus indicating proper conformation of this recombinant form. Clin Orthop, 2004 Jan, (418), 246 - 52 The effects of storage on fresh human osteochondral allografts; Ball ST et al.; Historically, fresh human osteochondral allografts have been stored in lactated Ringer's solution at 4 degrees C and then transplanted as quickly as possible, generally within 2 to 5 days, to ensure delivery of a high level of viable chondrocytes . Recently, allograft distribution companies have begun to provide fresh osteochondral allografts that are stored in a proprietary culture medium usually for at least 2 weeks before delivery to the surgeon for implantation . The effects of such storage on human cartilage have not been well-defined . In the current study the effects of storage in lactated Ringer's solution and in culture media were assessed . After 7 days of storage in lactated Ringer's solution, a significant decline in chondrocyte viability and metabolic activity was seen . Culture media provided significantly better preservation of the cartilage with viability and metabolic activity remaining essentially unchanged from baseline for as many as 14 days . The biochemical and biomechanical properties of the extracellular matrix remained stable with storage in both solutions with time . These data suggest that osteochondral allografts stored under traditional conditions in lactated Ringer's solution should continue to be implanted as quickly as possible and certainly within 7 days of donor death . If kept in culture media, the storage duration may be extended to approximately 2 weeks. Mol Reprod Dev, 2004 May, 68(1), 72 - 80 Differential growth, cell proliferation, and apoptosis of mouse embryo in various culture media and in coculture; Xu JS et al.; Sequential culture and coculture are two methods of improving the development of preimplantation embryos in vitro . Direct comparison of the efficiency of these methods is limited . Proliferation and apoptosis determine the total number of blastomere in preimplantation embryo, which is a sensitive parameter for evaluation of the development of embryo in vitro . In this study, we compared the proliferation and apoptosis of mouse embryo in different culture media, including CZB, KSOM, MTF, G1.2/G2.2 sequential culture media, and in human oviductal cell coculture . Sequential culture using G1.2/G2.2 was superior to KSOM, MTF, and CZB/CZB + G with respect to the formation of 3-4 cell embryos, morula, and blastocyst . G1.2/G2.2 cultured blastocyst had significantly more proliferating blastomeres and higher total cell number per blastocyst than those cultured in KSOM or CZB/CZB + G . Compared to embryos cultured in G1.2/G2.2, embryos cocultured in G1.2/G2.2 hatched more frequently . Cocultured blastocysts also had significantly higher percentage of proliferating cell and lower percentage of apoptotic cell per blastocyst than those cultured in G1.2/G2.2 . It was concluded that G1.2/G2.2 facilitated the proliferation of blastomere whilst human oviductal cell coculture suppressed apoptosis in addition to stimulating proliferation of blastomere . Protein Expr Purif, 2004 May, 35(1), 39 - 45 Optimized expression and refolding of human keratoepithelin in BL21 (DE3); Yuan C et al.; Keratoepithelin (KE) is an extracellular protein participating in cell adhesion and differentiation . Mutations of the KE gene (on 5q31 in humans) cause deposition of abnormal proteins (amyloid and non-amyloid) in corneal stroma and lead to several corneal dystrophies in humans . However, further studies on the KE protein have been limited by the intrinsic difficulty of purifying this protein . A high-expression plasmid containing human KE gene was constructed to generate recombinant KE proteins in Escherichia coli . The plasmid was transformed into E . coli BL21 (DE3) and the recombinant protein was expressed as an insoluble His-tagged fusion protein and purified by nickel chelation affinity chromatography under denaturing conditions . On average, 12 mg of purified KE was routinely obtained from 1L of culture media . The recombinant KE was refolded in arginine-containing dialysis solutions and the recovery of bioactive KE typically was approximately 70% . The procedures developed in this report should enable reproducible production of KE and related mutant proteins in large quantities and facilitate future studies on biochemical and biophysical properties of KE and the pathogenesis of related corneal dystrophies. Theriogenology, 2004 Apr 15, 61(6), 1125 - 35 In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres; Im GS et al.; This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos . Oocytes derived from a local abattoir were matured for 42-44 h and enucleated . Fetal fibroblasts were prepared from a Day 35 porcine fetus . Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes . Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium . For parthenogenetic activation, the same pulses were used . In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3) . Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23 . The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2) . In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4) . Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively) . However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ) . In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) . Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively . NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively . NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05) . Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05) . In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23 . Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23. Cancer Biol Ther, 2004 Mar, 3(3), 276 - 83 Epub 2004 Mar 08. TGFbeta1, back to the future: revisiting its role as a transforming growth factor; Glick AB; TGFbeta1 was initially identified in culture media from transformed cells as part of a factor that could produce a transformed phenotype in a nontransformed cell line . Subsequently this activity was separated into TGFbeta and TGFalpha an EGF receptor ligand . With the discovery that TGFbeta1 was a potent growth inhibitor of epithelial cells, and the identification of inactivating mutations within the TGFbeta1 signaling pathway in cancers it became clear that TGFbeta1 signaling is a tumor suppressor pathway for early stages of cancer . However many human carcinomas overexpress TGFbeta1 and this is associated with poor patient prognosis and increased frequency of metastasis . Similar results have been obtained with tumor cell lines and experimental animal models . Thus stage specific duality of function is the emerging paradigm for the role of TGFbeta1 in cancer . This review will focus on the evidence for TGFbeta1 as a tumor promoting and metastasis factor and examine the biological and molecular basis for these effects . It is proposed that the switch from tumor suppressor to oncogene reflects genetic or epigenetic alterations in signaling pathways in tumor cells that alter the readout from the TGFbeta1 pathway. J Biol Chem, 2004 May 14, 279(20), 21431 - 8 Epub 2004 Mar 19. Transthyretin, a new cryptic protease; Liz MA et al.; Transthyretin (TTR) is a plasma homotetrameric protein that acts physiologically as a transporter of thyroxine (T(4)) and retinol, in the latter case through binding to retinol-binding protein (RBP) . A fraction of plasma TTR is carried in high density lipoproteins by binding to apolipoprotein AI (apoA-I) . We further investigated the nature of the TTR-apoA-I interaction and found that TTR from different sources (recombinant and plasmatic) is able to process proteolytically apoA-I, cleaving its C terminus after Phe-225 . TTR-mediated proteolysis was inhibited by serine protease inhibitors (phenylmethanesulfonyl fluoride, Pefabloc, diisopropyl fluorophosphate, chymostatin, and N(alpha)-p-tosyl-l-phenylala-nine-chloromethyl ketone), suggesting a chymotrypsin-like activity . A fluorogenic substrate corresponding to an apoA-I fragment encompassing amino acid residues 223-228 (Abz-ESFKVS-EDDnp) was used to characterize the catalytic activity of TTR, including optimum reaction conditions (37 degrees C and pH 6.8) and catalytic constant (K(m) = 29 microm); when complexed with RBP, TTR activity was lost, whereas when complexed with T(4), only a slight decrease was observed . Cell lines expressing TTR were able to degrade Abz-ESFKVS-EDDnp 2-fold more efficiently than control cells lacking TTR expression; this effect was reversed by the presence of RBP in cell culture media, therefore proving a TTR-specific proteolytic activity . TTR can act as a novel plasma cryptic protease and might have a new, potentially important role under physiological and/or pathological conditions. Brain Res, 2004 Apr 9, 1004(1-2), 188 - 92 Glucose insufficiency alters neuronal viability and increases susceptibility to glutamate toxicity; Ioudina M et al.; Complete glucose deprivation has been shown to induce neuronal apoptosis, but the effect of moderate glucose deprivation under normal and pathological conditions is not fully understood . We investigated the effect of a restricted supply of glucose on neuronal vulnerability to glutamate by assaying cellular ATP levels (cellular energy production), 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT) reduction (mitochondrial function), lactate dehydrogenase (LDH) release (cellular viability) and activation of caspase-3 (apoptosis) in rat hippocampal neurons cultured in media (1.7, 5 and 25 mM glucose) with or without 100 microM glutamate . Cellular ATP levels were significantly reduced in neurons cultured in 1.7 mM glucose, while addition of glutamate markedly lowered cellular ATP levels even at the normal glucose concentration . MTT reduction was also significantly inhibited by 1.7 mM glucose; however, unlike cellular ATP levels, glutamate inhibition of MTT reduction was glucose concentration dependent . The LDH assay suggested that neuronal survival declines with decreasing glucose concentration in media, and glutamate potentiates this effect . Since low glucose media caused a decrease in cellular ATP and cell viability, we investigated apoptosis-related changes in cultured neurons by examining activity of caspase-3 . Low glucose media (1.7 and 5 mM glucose) increased caspase-3 activity, and glutamate potentiated this effect . Our results suggest that a low glucose supply in culture media activates an apoptosis mediator and markedly increases susceptibility to glutamate toxicity . Thus, even moderate glucose deprivation could be a serious risk factor that potentiates the pathophysiological consequences of certain neurodegenerative diseases. Physiol Plant, 2004 Feb, 120(2), 319 - 327 Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein; Sun W et al.; Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development . Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants . Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor . Here, this hypothesis was tested and supported with the following experiments . First, tomato (Lycopersicon esculentum, cv . UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody . Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv . Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody . Third, fluorescence microscopy of plasmolysed, transgenic tobacco (Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads . Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody . Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane . Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling. Acta Histochem, 2004 Feb, 106(1), 55 - 64 Immunodetection of aromatase in mice with a partial deletion in the long arm of the Y chromosome; Kotula-Balak M et al.; Aromatization of androgens into estrogens is catalyzed by a microsomal enzyme, P450 aromatase . Males of the mouse strain B10.BR and its congenic mutant strain B10.BR-Ydel (with a partial deletion in the long arm of the Y chromosome) were used to identify the cellular source of estrogens within the testis . Immunocytochemistry was applied to localize aromatase in cultured Leydig cells, cytoplasmic droplets attached to flagella of spermatozoa, and sections of testes . The presence of aromatase in testes was checked by means of Western-blot analysis . Steroid hormones secreted by Leydig cells in vitro were measured in homogenates of testes using radioimmunological methods . Additionally, a Southern analysis was performed using the Y353/B probe to check the length of the deletion in the Y chromosome . In sections of testis of B10.BR mice, weak to moderate immunohistochemical staining of aromatase was found in Leydig cells, Sertoli cells, and germ cells . In testicular cells of B10.BR-Ydel mice, stronger immunostaining of aromatase was observed, especially in germ cells and Leydig cells . Positivity for aromatase was also found in the cytoplasm of cultured Leydig cells from both strains, but it was higher in cells derived from mutant males . Western-blot analysis revealed one major band of approx . 55kDa of aromatase in testes from both strains . Lower testosterone levels were found in mutant males in supernatants of culture media and homogenates of testes in comparison with control males . In contrast, estradiol levels were always higher in mutants . Therefore, it seems likely that the increased expression of aromatase and, as a consequence, the higher levels of endogenous estrogens enhance the morphological alterations in testis and affect spermatogenesis in mutant males. Ann N Y Acad Sci, 2003 Dec, 1009, 20 - 9 Regulation of inducible nitric oxide synthase and agmatine synthesis in macrophages and astrocytes; Regunathan S et al.; Agmatine is a novel endogenous guanido amine synthesized from arginine by arginine decarboxylase . Among several biologic effects, the ability of agmatine to protect against ischemic injury and chronic neuropathic pain is particularly interesting . Because inflammation is a common contributor to these conditions, we sought to determine if agmatine acts by decreasing the production of proinflammatory molecules such as nitric oxide and if agmatine synthesis is regulated by inflammatory stimuli . We tested whether agmatine affects astroglial and macrophage (RAW 264.7 cell line) nitric oxide synthase-2 (NOS-2) expression . NOS-2 was induced in these cells by incubation with lipopolysaccharide (LPS) plus three cytokines for astrocytes and LPS alone for RAW 264.7 cells in the presence and absence of varying concentrations of agmatine . NOS-2 activity was assessed after 24 hours by nitrite accumulation in the culture media . Agmatine dose-dependently inhibited nitrite accumulation, and shorter incubation with agmatine (1 and 4 hours) also caused significant reduction . Agmatine decreased the expression of NOS-2 activity and NOS-2 protein as determined by immunoblot analysis . Incubation of astrocytes and RAW 264.7 cells with LPS/cytokines for 2 hours resulted in an increase in arginine decarboxylase (ADC) activity, whereas longer-term incubation (12-17 hours) lowered ADC activity . Agmatine levels in these cells are increased after 6-hour incubation with LPS/cytokines . These results show that agmatine inhibits the production of nitric oxide by decreasing the activity of NOS-2 in macrophages and astroglial cells by decreasing the levels of NOS-2 protein . These findings provide a molecular basis for the neuroprotective and anti-inflammatory actions of agmatine. Chem Res Toxicol, 2004 Mar, 17(3), 446 - 52 Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity; Carriere M et al.; Uranium is a naturally occurring heavy metal . Its extensive use in the nuclear cycle and for military applications has focused attention on its potential health effects . Acute exposures to uranium are toxic to the kidneys where they mainly cause damage to proximal tubular epithelium . The purpose of this study was to investigate the biological consequences of acute in vitro uranyl exposure and the influence of uranyl speciation on its cytotoxicity . NRK-52E cells, representative of rat kidney proximal epithelium, were exposed to uranyl-carbonate and -citrate complexes, which are the major complexes transiting through renal tubules after acute in vivo contamination . Before NRK-52E cell exposure, these complexes were diluted in classical or modified cell culture media, which can possibly modify uranyl speciation . In these conditions, uranium cytotoxicity appears after 16 h of exposure . The CI50 cytotoxicity index, the uranium concentration leading to 50% dead cells after 24 h of exposure, is 500 microM (+/-100 microM) and strongly depends on uranyl counterion and cell culture medium composition . Computer modeling of uranyl speciation is reported, enabling one to draw a parallel between uranyl speciation and its cytotoxicity. Exp Cell Res, 2004 Apr 1, 294(2), 550 - 8 Extracellular alpha 6 integrin cleavage by urokinase-type plasminogen activator in human prostate cancer; Demetriou MC et al.; During human prostate cancer progression, the integrin alpha6beta1 (laminin receptor) is expressed on the cancer cell surface during invasion and in lymph node metastases . We previously identified a novel structural variant of the alpha6 integrin called alpha6p . This variant was produced on the cell surface and was missing the beta-barrel extracellular domain . Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen activator (uPA) function-blocking antibody (3689), we showed that uPA, acting as a protease, is responsible for production of alpha6p . We also showed that addition of uPA in the culture media of cells that do not produce alpha6p, resulted in a dose-dependent alpha6p production . In contrast, the addition of uPA did not result in the cleavage of other integrins . Using alpha2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly . Further, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not alpha2-antiplasmin . Finally, the alpha6p integrin variant was detected in invasive human prostate carcinoma tissue indicating that this is not a tissue culture phenomenon . These data, taken together, suggest that this is a novel function of uPA, that is, to remove the beta-barrel ligand-binding domain of the integrin while preserving its heterodimer association. J Nutr Biochem, 2004 Mar, 15(3), 179 - 84 Copper may interact with selenite extracellularly in cultured HT-29 cells; Zeng H et al.; Previous studies have demonstrated that copper (15.7 micromol/L) can inhibit selenite (12.6 micromol/L)-induced cytotoxicity and apoptosis in HT-29 cells . However, the exact nature of the interactions between selenium and copper is not fully understood . In this study, the effect of copper on the cell cycle arrest induced by selenite or selenocystine was examined . Both selenite and selenocystine were effective in inhibition of cell growth and cell cycle progression . Cell cycle analysis revealed that selenite (3-5 micromol/L) caused a decrease in G1 phase cells that corresponded with an increase in S and G2 phase cells, and that 0.625 or 1.25 micromol/L copper sufficiently inhibited selenite-induced cell cycle arrest . In contrast, selenocystine caused an increase in G1 phase cells that corresponded with a decrease in S and G2 phase cells . Interestingly, 0.625 or 1.25 micromol/L copper did not inhibit selenocystine-induced cell cycle arrest . In addition, cell free gel shift assay demonstrated that selenite suppressed the inhibitory effect of copper on SP-1 DNA binding . Furthermore, although 5 micromol/L selenite in culture media significantly increased the intracellular selenium content, 1.25 micromol/L copper sulfate blocked this increase of the intracellular selenium content . Collectively, these data demonstrate that selenite and selenocystine cause cell cycle arrest via distinct mechanisms, and suggest that copper may interact with selenite extracellularly, which represents the basis of antagonism between copper sulfate and selenite. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Feb, 21(1), 34 - 7 {Time-dependent increase of interleukin-8 production in endothelial cells exposed to fluid shear stress}; Tang R et al.; Fluid shear stress plays an important role in many physiological and pathological processes of the cardiovascular system . Being constantly exposed to mechanical shear stress, vascular endothelial cells can sense the changes of blood flow forces and regulate vascular structure and function . Previous studies demonstrated that IL-8 mRNA expression in endothelial cells was modulated by fluid shear stress . To identify the effect of fluid shear stress on IL-8 protein production of human umbilical vein endothelial cells (HUVECs), we employed quantitative sandwich enzyme-linked immunosorbent assay (ELISA) to measure the IL-8 protein . It was found that the HUVECs not treated with fluid shear stress secreted very little IL-8 in culture media . However, after 1 hour of exposure to shear stress, the secretion of IL-8 increased; at 5 hours of exposure, the seceretion reached the summit; at 8 hours of exposure, the secretion of IL-8 decreased and then remained at a constant level till the end (12 hours) of the experiment . The increase of IL-8 secretion induced by shear stress was time-dependent . The biphasic response of IL-8 protein production was found in experiments in which the shear stress applied was 2.09 dyne/cm2, 4.61 dyne/cm2, and 6.19 dyne/cm2 . The IL-8 protein production in response to shear stress was very similar to the IL-8 gene expression in response to shear stress, and had the obvious delay . The induction of IL-8 protein production by fluid shear stress is probably due to the gene expression . This in vitro study demonstrates that the production of IL-8 can be regulated by fluid shear stress . Fluid shear stress induces a biphasic response of human HUVECs' production of IL-8 protein . These observations suggest that the process of the fluid shear stress induced HUVECs' production of IL-8 may play an important role in the genesis and development of both inflammation and atherosclerosis. Pediatr Dev Pathol, 2004 Mar-Apr, 7(2), 148 - 58 Epub 2004 Mar 17. Cytogenetic distinction among benign fibro-osseous lesions of bone in children and adolescents: value of karyotypic findings in differential diagnosis; Parham DM et al.; Benign fibro-osseous lesions of bone (BFOL) comprise a group of clinically distinct entities with significant histologic overlap and often occur in children and adolescents . Because of prior studies indicating that these lesions possess distinct karyotypic abnormalities, we conducted a retrospective review of cytogenetic analyses performed in a series of 16 BFOL in children and adolescents diagnosed at two institutions . These comprised five cases with the diagnosis of ossifying fibroma, four with osteofibrous dysplasia, and seven with fibrous dysplasia arising in the skeleton of 16 children and adolescents . All cases were analyzed using standard G-banding techniques on fresh tumors explanted in tissue culture media . Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) were used to analyze selected metaphases of a talar lesion with the histologic features of ossifying fibroma . All four confirmed ossifying fibromas, including the talar lesion, contained clonal aberrations fusing breakpoints on Xq26 and 2q33, and one case with dissimilar histology did not . Three of four osteofibrous dysplasias contained multiple copies of chromosomes 8, 12, and/or 21 . All but two fibrous dysplasia cases exhibited either a completely normal karyotype or single cell aberrations . One fibrous dysplasia had subtle chromosomal abnormalities not seen in other cases in the series, and another had complex abnormalities involving multiple chromosomes . Our current and published results indicate that cytogenetics might be of ancillary use in the diagnosis of BFOL and that a characteristic chromosomal arrangement is associated with ossifying fibroma. Methods Mol Biol, 2004, 264, 245 - 57 ProteinChip array-based amyloid beta assays; Bradbury LE et al.; Amyloid-beta (A beta) fragments are found in plaques of patients with Alzheimers . Three secretases cleave the amyloid precursor protein, producing multiple A beta fragments that accumulate in the brain and fluids of patients with Alzheimers . A beta peptides are difficult to detect using standard methods because of their small size and multiple isoforms . However, multiple peptide fragments can be detected using a single ProteinChip Array-Based assay . Specific antibodies recognizing various amyloid epitopes are immobilized on a ProteinChip Array . Crude samples, such as tissue lysates, serum, cerebral spinal fluid (CSF), or cell culture media, are applied to the antibody-coated arrays . A beta peptides are specifically retained by the antibody, whereas other sample components are removed by washing . The multiple peptide fragments are detected by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), which can easily resolve the different fragments because of the corresponding changes in peptide mass. J Cell Sci, 2004 Mar 15, 117(Pt 8), 1567 - 76 Trophic signals acting via phosphatidylinositol-3 kinase are required for normal pre-implantation mouse embryo development; Lu DP et al.; The growth and survival of the preimplantation mammalian embryo may be regulated by several autocrine trophic factors that have redundant or overlapping actions . One of the earliest trophic factors to be produced is embryo-derived platelet-activating factor (1-O-alky-2-acetyl-sn-glyceryl-3-phosphocholine) . The addition of platelet-activating factor to embryo culture media exerted a trophic effect, but structurally related lipids (3-O-alky-2-acetyl-sn-glyceryl-1-phosphocholine, 1-O-alky-sn-glyceryl-3-phosphocholine, octadecyl-phosphocholine) had no effect . Platelet-activating factor induced a pertussis toxin-sensitive {Ca(2+)}(i) transient in two-cell embryos that did not occur in platelet-activating factor-receptor null (Pafr-/-) genotype embryos . Fewer Pafr-/- mouse zygotes developed to the blastocyst stage in vitro compared with Pafr+/+ zygotes (P<0.02), those that developed to blastocysts had fewer cells (P<0.001) and more cells with fragmented nuclei (P<0.001) . The inhibition of 1-O-phosphatidylinositol 3-kinase (LY294002 (3 microM and 15 microM) and wortmannin (10 nM and 50 nM)) caused a dose-dependent inhibition of platelet-activating factor-induced {Ca(2+)}(i) transients (P<0.001) . The two-cell embryo expressed 1-O-phosphatidylinositol 3-kinase catalytic subunits p110 alpha, beta, gamma and delta, and regulatory subunits p85 alpha and beta . LY294002 and wortmannin each caused a significant reduction in the proportion of embryos developing to the morula and blastocyst stages in vitro, reduced the number of cells within each blastocyst, and significantly increased the proportion of cells in blastocysts with fragmented nuclei . The results indicate that embryo-derived platelet-activating factor (and other embryotrophic factors) act through its membrane receptor to enhance embryo survival through a 1-O-phosphatidylinositol 3-kinase-dependent survival pathway. Sci Total Environ, 2004 Mar 29, 320(2-3), 259 - 67 Arsenic oxidation and bioaccumulation by the acidophilic protozoan, Euglena mutabilis, in acid mine drainage (Carnoulès, France); Casiot C et al.; In the acid stream (pH 2.5-4.7) originating from the Carnoules mine tailings, the acidophilic protozoan Euglena mutabilis grows with extremely high sulfate (1.9-4.9 g/l), iron (0.7-1.7 g/l) and arsenic concentrations (0.08-0.26 g/l) . Strong variations in flow rate and high sulfate concentrations (up to 4.9 g/l) have been registered in early winter and might be the reason for the reduction in cell number of the protozoan from October to December 2001 . No relation was established between arsenic concentration and/or speciation and abundance of the protozoan in the stream . Arsenite, which is the most toxic form, predominates in water . The oxidation of arsenite to arsenate occurred within a few days in laboratory experiments when E . mutabilis was present in Reigous Creek water and synthetic As(III)-rich culture medium . Methylated compounds (MMA, DMA) were not identified in the culture media . The protozoan bioaccumulated As in the cell (336 +/- 112 microg As/g dry wt.) as inorganic arsenite (105 +/- 52 microg As/g dry wt.) and arsenate (231 +/- 112 microg As/g dry wt.) . Adsorption of As at the cell surface reached 57 mg/g dry wt . in the As(V) form for E . mutabilis grown in 250 mg/l As(III) synthetic medium . Both intracellular accumulation and adsorption at the cell surface increased for increasing As(III) concentration in the medium but the concentration factor in the cell relative to soluble As decreased. J Huazhong Univ Sci Technolog Med Sci, 2003, 23(4), 375 - 9 Suramin inhibits the in vitro expression of encephalitis B virus proteins NS3 and E; Xu K et al.; In this study, the mechanism by which Suramin inhibits the replication of epidemic encephalitis B virus was explored to provide a theoretical basis for its further application in clinical practice . After viral infection of HepG2 and IMR-32 cells, different concentrations of Suramin were added to the culture media, and then the cultural supernatants and infected cells were collected 48 h later . For the evaluation of the curative effect, cytopathic effect (CPE), virus titers, the expression of viral protein and viral RNA were determined by Western blot, RT-PCR and in vitro RNA synthesis, respectively . At the concentration of 50 microg/ml of Suramin, HepG2 and IMR-32 infected with epidemic encephalitis B virus decreased by 51.8% and 0.03% respectively, as compared with controls . It was suggested that expression of encephalitis B virus proteins NS3 and E was notably reduced by Suramin . This is especially true of E protein . At RNA level, however, no difference in RNA virus was found between Suramin-treated virus and non-treated cells . Our results suggest that Suramin can inhibit viral replication by blocking the production of viral proteins. Zhongguo Zhong Yao Za Zhi, 2003 Jun, 28(6), 536 - 40 {Establishment of drug screening model based on transcriptional regulation of estrogen responsive element}; Wang LQ et al.; OBJECTIVE: AIM To establish a drug screening model based on transcriptional regulation of estrogen responsive element (ERE) and use it to screen compounds for discovering new ligands of estrogen receptor (ER) subtypes . METHOD: A recombinant reporter vector pERE-TAL-SEAP was constructed by inserting a synthetic sequence composed of five tandem copies of EREs upstream of promoter of the reporter vector pTAL-SEAP . The pERE-TAL-SEAP and the internal control plasmid pCMV were transiently co-transfected into Hela cells expressing ER subtype or ER subtype, and the effects of pure ER agonists 17estradiol, phytoestrogen genistein and pure ER antagonist ICI182, 780 on reporter gene SEAP expression were observed . RESULT: In the Hela cells expressing ER alpha or ER beta subtype, the expression of SEAP gene were induced in a dose dependent manner by 17-estrodiol with a maximal effect at approximately 10 nmol.L-1 and with EC50 of (80.58 +/- 8.51) pmol.L-1 and (103.90 +/- 5.29) pmol.L-1, respectively, so done by phytoestrogen genistein with a maximal effect at 1 mumol.L-1 and with EC50 of (10.86 +/- 0.75) nmol.L-1 and (39.38 +/- 2.26) nmol.L-1, respectively . The maximal level induced by estrodiol and genistein were about 7-14 fold higher than that of vehicle . The pure antiestrogen ICI182, 780 at concentration of 1 mumol.L-1 completely blocked the inductions of 17-estrodiol and genistein . CONCLUSION: The cellular drug screening model can be established by transfecting reporter vector pERE-TAL-SEAP in Hela cell lines expressing ER alpha or ER beta . The cell lines can be used to screen compounds with estrogenicity by testing SEAP activity in the culture media of cells growing in microtitier wells . The system should provide an efficient model for screening and analyzing the activity of large numbers of ligands of ER. In Vivo, 2004 Jan-Feb, 18(1), 43 - 7 Glucocorticoid receptor function suppresses insulin-like growth factor 1 activity in human KLE endometrial-like cells; Lembessis P et al.; We analysed the glucocorticoid receptor (GR) regulation on the expression of insulin-like growth factor 1 (IGF-1), type I IGF receptor (IGF-1.R), IGF-binding protein 3 (IGFBP-3), urokinase-type plasminogen activator (uPA) and uPA receptor (uPA.R) mRNA in human KLE endometrial-like cells . We documented that KLE cells express IGF-1, IGF-1.R, uPA and IGFBP-3 mRNA, however not uPA.R mRNA . Exogenous administration of dexamethasone inhibited the proliferation of KLE cells without inducing apoptosis . The inhibition of dexamethasone on KLE cell proliferation was neutralized by exogenous administration of IGF-1 . Furthermore, dexamethasone suppressed the expression of IGF-1 mRNA and IGF-1.R mRNA as well as the IGF-1 bioavailability in KLE cell culture media, but it did not alter the expression of uPA mRNA and IGFBP-3 mRNA in KLE cells . Since the peritoneal fluid of women with endometriosis is known to contain IGF-1, which stimulates the proliferation and inhibits the apoptosis of endometrial-like cells, it is conceivable that GR-mediated down-regulation of IGF-1 bioavailability may be of clinical relevance for endometriosis. Tissue Eng, 2004 Jan-Feb, 10(1-2), 139 - 44 Expansion of the number of human auricular chondrocytes: recycling of culture media containing floating cells; Kamil SH et al.; To grow a complete human size auricle by utilizing the principles of tissue engineering, a large number of chondrocytes is required for initial implantation . The number of chondrocytes can be increased by repeated passaging or by incubation with different growth factors, both of which can promote dedifferentiation . New methods of chondrocyte expansion over a relatively brief time period for potential practical application are required . In this study auricular chondrocytes were obtained from patients and cultured in vitro . Two groups of cells were created . Group A chondrocyte number was increased by repeated passaging . Group B cells were grown from floating culture medium and their number was increased both by passaging and by repeated recycling of the culture medium . Chondrocytes from both groups were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage . Flow cytometry studies performed on both groups confirmed the presence of two distinct populations of structures as the source of chondrocytes from the recycled medium . Repeated recycling of the culture medium demonstrates a promising method to increase the number of chondrocytes in vitro for clinical application. Hypertension, 2004 Apr, 43(4), 866 - 71 Epub 2004 Mar 08. Sgk1 mediates osmotic induction of NPR-A gene in rat inner medullary collecting duct cells; Chen S et al.; We have shown previously that increased extracellular osmolality stimulates expression and promoter activity of the type A natriuretic peptide receptor (NPR-A) gene in rat inner medullary collecting duct (IMCD) cells through a mechanism that involves activation of p38 mitogen-activated protein kinase (MAPK) . The serum and glucocorticoid inducible kinase (Sgk) is thought to participate in the regulation of sodium handling in distal tubular segments . We sought to determine whether this kinase might be involved in the osmotic stimulation of NPR-A gene promoter activity . Exposure of cultured IMCD cells to an additional 75 mmol/L NaCl in culture media (final osmolality 475 mosm/kg) resulted in an approximately 4-fold increase in Sgk1 protein levels after 7 hours . The Sgk1 induction was almost completely inhibited by the p38 MAPK inhibitor SB203580, indicating that NaCl activates Sgk1 through the p38 MAPK pathway . Transient transfection of a mouse Sgk1 expression vector along with a -1590 NPR-A luciferase reporter resulted in an approximately 3-fold increment in reporter activity, which was significantly reduced by cotransfection with a kinase-dead Sgk1 mutant . The NaCl-dependent induction was partially blocked (approximately 40% inhibition) by cotransfection of the kinase-dead Sgk1 mutant . Neither Sgk1 nor the kinase-dead mutant had any effect on endothelial nitric oxide synthase (eNOS) promoter activity, and the Sgk1 mutant and 8-bromo-cyclic guanosine monophosphate were, to some degree, additive in reducing osmotically stimulated NPR-A promoter activity . Collectively, these data imply that Sgk1 operates over an eNOS-independent, p38 MAPK-dependent pathway in mediating osmotic induction of the NPR-A gene promoter. Tumour Biol, 2003 Sep-Oct, 24(5), 219 - 27 Transient increase in squamous cell carcinoma antigen expression in cultured cervical carcinoma CaSki cells in response to exposure to cis-diamminedichloroplatinum; Tateiwa Y et al.; The present study was conducted to elucidate the molecular mechanism underlying the transient increase in circulating squamous cell carcinoma antigen (SCC Ag) levels in response to CIS-diamminedichloroplatinum (CDDP) infusion using an in vitro model . The uterine cervical squamous carcinoma CaSki cells were cultured for 72 h after 3 h exposure to 5.0 microg/ml CDDP . The effects of CDDP exposure on the proliferative activity and apoptosis in cultured CaSki cells were determined by bromodeoxyuridine (BrdU) uptake and cell counting and by the TUNEL assay, respectively . SCC Ag levels in cultured CaSki cells and culture media were determined with the use of SCC-RIA kit . The expression of SCC Ag-1 mRNA and SCC Ag-2 mRNA in cultured CaSki cells was assessed using semiquantitative RT-PCR with Southern blot analysis . The number of BrdU-positive CaSki cells significantly decreased 6 h after exposure to CDDP, whereas the apoptosis-positive rate of cultured CaSki cells significantly increased 12 h after the CDDP exposure . The number of cultured CaSki cells significantly decreased 72 h after the CDDP exposure . The total SCC Ag protein levels in both cultured CaSki cells and the culture media after the 3-hour CDDP exposure increased in a time-dependent manner during the subsequent incubation for 48 h . Semiquantitative RT-PCR revealed that the expression levels of both SCC Ag-1 and SCC Ag-2 mRNA increased (1.7- and 2.7-fold, respectively) 12 h after the exposure to CDDP relative to those before the subsequent cultures . Exposure of uterine cervical squamous carcinoma CaSki cells to CDDP resulted in a transient increase in SCC Ag protein and mRNA expression in those cells during the initial 12 h after the exposure, being associated with decreased proliferative activity and increased apoptosis of those cells . Anim Reprod Sci, 2004 Apr, 81(3-4), 273 - 86 Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water; Silva JR et al.; The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis . We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex . The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100% . The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA . Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls . The replicates were the two ovaries of five mixed breed goats . The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated . Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA) . The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test . Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells . As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles . The fall in the number of primordial follicles was particularly marked after 1 day culture . No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles . Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles . In contrast, addition of supplements to culture media reduced follicular degeneration . In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles . This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture . In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested . However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration. Br J Cancer, 2004 Mar 8, 90(5), 1100 - 7 Selective disruption of the E-cadherin-catenin system by an algal toxin; Ronzitti G et al.; Yessotoxins (YTXs) are algal toxins that can be accumulated in edible molluscs . YTX treatment of MCF-7 breast cancer cells causes the accumulation of a 100 kDa fragment of E-cadherin, which we have named ECRA(100) . A relative decrease in the concentrations of intact E-cadherin did not accompany the accumulation of ECRA(100) in cytosoluble extracts of MCF-7 cells on the first day of YTX treatment, but a collapse of the E-cadherin system was detected after 2-5 days of treatment with the toxin . An analysis of the general structure of ECRA(100) revealed that it consists of an E-cadherin fragment lacking the intracellular domain of the protein . ECRA(100) was not released into culture media of YTX-treated cells . Accumulation of ECRA(100) was observed in other epithelial cells, such as human intestine Caco-2 and MDCK cells after treatment with YTX . In turn, YTX could not induce accumulation of fragments of other members of the cadherin family, such as N-cadherin in the PC12 cell line and K-cadherin in sensitive cells (MCF-7, Caco-2, MDCK) . The accumulation of a 100 kDa fragment of E-cadherin devoid of its intracellular domain induced by YTX was accompanied by reduced levels of beta- and gamma-catenins bound to E-cadherin, without a concomitant decrease in the total cytosoluble pools of beta- and gamma-catenins . Taken together, the results we obtained show that YTX causes the selective disruption of the E-cadherin-catenin system in epithelial cells, and raise some concern about the potential that an algal toxin found in seafood might disrupt the tumour suppressive functions of E-cadherin. J Bacteriol, 2004 Mar, 186(6), 1667 - 77 Phosphatidylethanolamine is not essential for growth of Sinorhizobium meliloti on complex culture media; Sohlenkamp C et al.; In addition to phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE), Sinorhizobium meliloti also possesses phosphatidylcholine (PC) as a major membrane lipid . The biosynthesis of PC in S . meliloti can occur via two different routes, either via the phospholipid N-methylation pathway, in which PE is methylated three times in order to obtain PC, or via the phosphatidylcholine synthase (Pcs) pathway, in which choline is condensed with CDP-diacylglycerol to obtain PC directly . Therefore, for S . meliloti, PC biosynthesis can occur via PE as an intermediate or via a pathway that is independent of PE, offering the opportunity to uncouple PC biosynthesis from PE biosynthesis . In this study, we investigated the first step of PE biosynthesis in S . meliloti catalyzed by phosphatidylserine synthase (PssA) . A sinorhizobial mutant lacking PE was complemented with an S . meliloti gene bank, and the complementing DNA was sequenced . The gene coding for the sinorhizobial phosphatidylserine synthase was identified, and it belongs to the type II phosphatidylserine synthases . Inactivation of the sinorhizobial pssA gene leads to the inability to form PE, and such a mutant shows a greater requirement for bivalent cations than the wild type . A sinorhizobial PssA-deficient mutant possesses only PG, CL, and PC as major membrane lipids after growth on complex medium, but it grows nearly as well as the wild type under such conditions . On minimal medium, however, the PE-deficient mutant shows a drastic growth phenotype that can only partly be rescued by choline supplementation . Therefore, although choline permits Pcs-dependent PC formation in the mutant, it does not restore wild-type-like growth in minimal medium, suggesting that it is not only the lack of PC that leads to this drastic growth phenotype. J Viral Hepat, 2004 Mar, 11(2), 124 - 9 Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells; Liu MC et al.; The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009) . The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644) . We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA . HBV cccDNA was found both intracellularly and extracellularly . A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively) . These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents. Eur J Nutr, 2004 Feb, 43(1), 23 - 31 Epub 2004 Jan 06. Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells; Crisp SE et al.; BACKGROUND: Placental transfer of nutrients and secretion of hormones is essential for normal fetal development . AIM OF THE STUDY: To determine whether biotin supply affects biotin homeostasis, proliferation rates, and progesterone secretion in placenta cells . METHODS: JAr choriocarcinoma cells were cultured in media containing deficient (25 pmol/L), physiological (250 pmol/L), or pharmacological concentrations (10,000 pmol/L) of biotin for three weeks; markers for biotin homeostasis, proliferation, and hormone secretion were quantified . RESULTS: Biotin concentrations in culture media correlated negatively with expression of the biotin transporter SMVT, as judged by cellular transport rates of biotin, abundance of SMVT protein, and transcriptional activity of SMVT reporter-gene constructs . Notwithstanding this homeostatic mechanism, biotin concentrations in media correlated positively with activities of biotin-dependent propionyl-CoA carboxylase, abundance of biotinylated carboxylases, and with biotinylation of histones . Biotin deficiency was associated with decreased rates of thymidine uptake into JAr cells {pmol thymidine/( 10(6) cells x 24 h)}: 1.6 +/- 0.1 (25 pmol/L biotin) versus 2.3 +/- 0.2 (250 pmol/L biotin) versus 3.7 +/- 0.4 (10,000 pmol/L biotin), suggesting that cell proliferation depends on biotin . Secretion of progesterone was reduced in biotin-deficient cells; this effect was caused by reduced generation of new cells in deficient media rather than by an immediate effect of biotin on progesterone secretion at the singlecell-level . CONCLUSIONS: This study provides evidence that choriocarcinoma cells cannot maintain normal activities of biotin-dependent metabolic pathways if biotin concentrations in culture media are low . It is uncertain whether activities of biotin-dependent pathways in placenta affect fetal development in vivo. J Virol, 2004 Mar, 78(6), 2819 - 30 Retinoid-dependent restriction of human immunodeficiency virus type 1 replication in monocytes/macrophages; Hanley TM et al.; Vitamin A deficiency has been correlated with increased severity of human immunodeficiency virus type 1 (HIV-1)-associated disease . Moreover, vitamin A supplementation can reduce AIDS-associated morbidity and mortality . Our group and others have shown that retinoids, the bioactive metabolites of vitamin A, repress HIV-1 replication in monocytic cell lines and primary macrophages by blocking long-terminal-repeat (LTR)-directed transcription . Based on these studies, we hypothesize that retinoids are natural repressors of HIV-1 in vivo . We show here that all-trans-retinoic acid (RA)-mediated repression of HIV-1 activation requires pretreatment for at least 12 h and is blocked by the protein synthesis inhibitors cycloheximide and puromycin . Studies of the kinetics of RA-mediated repression in U1 cells and primary monocyte-derived macrophages (MDMs) reveal that the repressive effects of RA on HIV-1 expression are long-lasting but reversible . We demonstrate that HIV-1 expression is activated when U1 cells or MDMs are cultured in retinoid-free synthetic medium and show that physiological concentrations of RA repress this activation . In addition, the synthetic pan-retinoic acid receptor antagonist BMS-204 493 activates HIV-1 replication in U1 cells in a dose-dependent manner, suggesting that RA-induced transactivation of cellular gene expression is required for HIV-1 repression . Together, these data support the hypothesis that retinoids present in tissue culture media in vitro and serum in vivo maintain HIV-1 in a transcriptionally repressed state in monocytes/macrophages. Hum Reprod, 2004 Apr, 19(4), 940 - 7 Epub 2004 Feb 27. Frequent structural chromosome aberrations in immotile human sperm exposed to culture media; Watanabe S; BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes . METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes . In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection . In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection . In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes . The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically . RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%) . In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%) . In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead . In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%) . CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media. Zhong Yao Cai, 2003 Nov, 26(11), 795 - 8 {Inhibitive effect of curcumin and amiloride on the fibrosis of rat hepatic stellate cells induced by oxidative stress}; Yang W et al.; OBJECTIVE: To investigate the effects of antioxidant curcumin and Na+/H+ exchanger inhibitor amiloride on the fibrosis of rat hepatic stellate cells (HSC) induced by oxidative stress in vitro . METHODS: HSC were incubated with 0.1 mmol/L ferric nitrilotriacetate complex (FeNTA) . MTT colorimetry was used for assaying proliferation of HSC . Cytotoxicity was measured by LDH colorimetry . Collagen type I accummulation in the culture media was measured by ELISA . Intracellular malonildialdehyde (MDA), SOD, GSH and GSH-Px levels in the culture media were measured by respective reagent boxes . RESULTS: HSC incubation with FeNTA resulted in a significant production of intracellular MDA and GSH, associated with decrease of SOD and GSH-PX activity . Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium . All these effects were reversed by the antioxidant curcumin and by the Na+/H+ exchanger inhibitor amiloride . Combining curcumin with amiloride had better effects on the fibrosis of rat hepatic stellate cells induced by oxidative stress than curcumin or amiloride . CONCLUSION: The effects of combination of antifibrosis drugs curcumin and amiloride on different targets of HSC are superior to curcumin or amiloride. FEMS Microbiol Lett, 2004 Feb 16, 231(2), 237 - 45 Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis; Malhotra V et al.; The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory . A devR::kan mutant of M . tuberculosis was constructed by allelic exchange . The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media . This phenotype was reversed on complementation with a wild-type copy of devR . The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells . The expression of DevR was not modulated upon entry of M . tuberculosis into human monocytes . However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain . Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M . tuberculosis within human monocytes in vitro . The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M . tuberculosis within tissues. Horm Metab Res, 2004 Jan, 36(1), 7 - 13 Retinoic acid increases insulin-like growth factor-binding protein-4 expression in cultured rat hepatocytes; Demori I et al.; Hepatic insulin-like growth factor binding protein (IGFBP) expression is controlled by diverse factors including thyroid hormone, which enhances IGFBP-4 production in hepatocytes . In the present work, we have investigated whether hepatic IGFBP-4 expression is regulated by retinoic acid (RA), which acts via nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily . Primary cultures of adult rat hepatocytes were incubated with two natural stereoisomers of RA, all-trans RA and 9-cis RA (atRA and 9cRA), and with the synthetic RA receptor (RAR)-selective agonist TTNPB . IGFBP-4 mRNA abundance was measured by Northern blot and protein production was evaluated by Ligand blot on hepatocyte-conditioned culture media . Our results indicate that atRA, 9cRA, and TTNPB increase IGFBP-4 expression by cultured hepatocytes, both at the mRNA and protein level . The RARs play a definite role in this regulation, which is independent from ongoing protein synthesis but dependent on active transcription . AtRA and thyroid hormone act synergistically in increasing hepatic IGFBP-4 expression . Our data establish a role for hormonal factors such as thyronines and retinoids in regulating the hepatic IGF system directly at the IGFBP-4 level. Anesth Analg, 2004 Mar, 98(3), 841 - 5, table of contents Intracellular calcium increases in growth cones exposed to tetracaine; Saito S et al.; Neurotoxicity of local anesthetics has been reported for both matured and growing neurons . In the present study, we examined if tetracaine increases Ca(2+) concentration during growth cone collapse . Intracellular Ca(2+) concentration was measured by fura 2/AM after exposure to tetracaine . Tetracaine (1-2 mM) induced increases in intra-growth cone Ca(2+) concentration (P < 0.01) . The Ca(2+) hot spot was expanded into the neurite from the periphery towards the cell body . When tetracaine was applied to growth cones in Ca(2+) free media, the increase was minor . However, tetracaine induced growth cone collapse even in the culture media, which did not contain Ca(2+) . Ni(2+) (100 microM; a general Ca(2+) channel inhibitor) and BAPTA-AM (5 microM; intracellular Ca(2+) chelator) could not inhibit growth cone collapse induced by 1-2 mM tetracaine . Tetracaine (>1 mM) induces collapse and Ca(2+) increase at growth cones simultaneously; however, these two phenomena might be provoked independently . IMPLICATIONS: Tetracaine induced intracellular Ca(2+) increases and growth cone collapse in dorsal root ganglion neurons . The Ca(2+) hot spot in the growth cone expanded into the neurite from periphery towards the cell body. Biomaterials, 2004 Jul, 25(16), 3187 - 99 The tensile properties of alginate hydrogels; Drury JL et al.; Alginate hydrogels are currently being employed and explored for a broad range of medical applications including cell encapsulation, drug delivery, and tissue engineering . In these capacities, knowledge of the mechanical and material properties of the hydrogels and the properties that govern and influence them is necessary to adequately design and effectively use these systems . Although much is known about the mechanical properties of alginate in compression and shear, little is known about the tensile characteristics . Thus, an extensive tensile assessment of alginate hydrogels was completed as a function of alginate type, formulation, gelling conditions, incubation, and strain rate . In general, the initial tensile behavior and properties of alginate hydrogels were highly dependent on the choice of the alginate polymer and how it was processed . Specifically, high guluronic acid containing alginate polymers yielded stronger, more ductile hydrogels than high mannuronic acid containing alginates . The ultimate stress, ultimate strain, and tensile modulus were decreased by increased phosphate concentrations, solution reconstitution with phosphate buffered saline instead of culture media, and peptide modification . Incubation of hydrogels for at least 7 days diminished many of the initial tensile property differences associated with formulation and gelling conditions . Overall, by controlling the specific alginate polymer and processing methods, a wide range of tensile properties are available from these hydrogels. J Am Soc Nephrol, 2004 Mar, 15(3), 575 - 84 Exogenous attenuation of p21(Waf1/Cip1) decreases mesangial cell hypertrophy as a result of hyperglycemia and IGF-1; Fan YP et al.; Renal mesangial cell hypertrophy is a characteristic of diabetic nephropathy as well as a response to renal stress or injury . Because hypertrophy is a result of increased protein content per cell without DNA replication, those proteins that control the cell cycle, such as the cyclin kinase inhibitor p21, represent fertile ground for studying the mechanism of this structural alteration . A key role for p21 in promoting mesangial cell (MC) hypertrophy has been established using p21 knockout mouse models . Furthermore, some of the biologic effects of IGF-1, including cell proliferation, have been shown to be positively influenced by p21 . In an attempt to begin to translate these findings ultimately to the bedside, methods to attenuate p21 levels in wild-type kidney cells were examined . With the use of a phosphorothioated antisense oligodeoxynucleotide (ODN) to p21, which has previously been shown to decrease specifically and effectively p21 protein levels in a variety of cell types, it is shown that attenuation of p21 in MC leads to a dose-dependent reduction of hypertrophy in the milieu of hyperglycemic culture media . Furthermore, the hypertrophic effect of the IGF-1 on MC is also attenuated using the same antisense p21 ODN . There was no evidence of apoptosis or other toxicity in MC transfected with the concentrations of antisense p21 ODN used in these experiments . Because the use of antisense ODN in human disease is already established in other medical disciplines, the stage is now set for the use of antisense p21 ODN to attenuate renal cell hypertrophy in vivo, leading to a new strategy for treatment of diabetic nephropathy and other diseases characterized by MC hypertrophy. Endocrinology, 2004 Jun, 145(6), 2978 - 87 Epub 2004 Feb 19. Transactivation of the insulin-like growth factor-I receptor by angiotensin II mediates downstream signaling from the angiotensin II type 1 receptor to phosphatidylinositol 3-kinase; Zahradka P et al.; Angiotensin II (AngII) activates phosphatidylinositol 3-kinase (PI3-kinase), a known effector of receptor tyrosine kinases . Treatment of smooth muscle cells with AngII has also been shown to promote phosphorylation of various tyrosine kinase receptors . We therefore investigated the relationship between AngII and IGF-I receptor activation in smooth muscle cells with a phosphorylation-specific antibody . Our experiments showed that IGF-I receptor phosphorylation was maximally stimulated within 10 min by AngII . Inclusion of an IGF-I-neutralizing antibody in the culture media did not prevent IGF-I receptor phosphorylation after AngII treatment, which argues that a paracrine/autocrine loop is not required . Furthermore, this process was blocked by losartan and 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo{3,4-d}pyrimidin-4-amine (PP-1), indicating stimulation of IGF-I receptor phosphorylation occurs via AngII type 1 receptor-dependent activation of Src kinase . The functional significance of IGF-I receptor transactivation was examined with selective inhibitors of the IGF-I receptor kinase (AG1024, AG538) . When AngII-treated cells were incubated with AG1024 or AG538, phosphorylation of the regulatory p85 subunit of PI3-kinase was blocked . Furthermore, phosphorylation of the downstream factor p70(S6K) did not occur . In contrast, AG1024 did not prevent MAPK or Src kinase activation by AngII . AG1024 also did not inhibit AngII-dependent cell migration, although this process was blocked by inhibitors of the epidermal growth factor and platelet-derived growth factor receptors . Transactivation of the IGF-I receptor is therefore a critical mediator of PI3-kinase activation by AngII but is not required for stimulation of the MAPK cascade. Reprod Fertil Dev, 2004, 16(2), 15 - 25 Developments in in vitro technologies for swine embryo production; Wheeler MB et al.; Several modifications have been made to in vitro production (IVP) systems to allow more efficient production of viable porcine embryos . Although in vitro production of pig embryos has been studied for over 30 years, the overall blastocyst production rate remains low . The low blastocyst rate is due to several factors, including polyspermic oocyte penetration, low rate of male pronucleus formation and less than optimal in vitro culture systems . These conditions are all inherent problems in porcine IVP and many of the mechanisms involved remain unknown . Considerable research has examined culture medium and the techniques used during the various stages of in vitro production . However, changes to the physical culture system used during IVF have remained unchanged until recently . The present paper will summarise selected developments in fertilisation and embryo culture media composition and focus on the development of modified equipment to improve the conditions used during the IVP of porcine oocytes and embryos. Arch Pharm Res, 2004 Jan, 27(1), 86 - 93 Involvement of nitric oxide during in vitro fertilization and early embryonic development in mice; Kim BH et al.; Nitric oxide (NO) has emerged as an important intracellular and intercellular messenger, controlling many physiological processes and participating in the fertilization process via the autocrine and paracrine mechanisms . This study investigated whether nitric oxide synthase (NOS) inhibitior (L-NAME) and L-arginine could regulate in vitro fertilization and early embryonic development in mice . Mouse epididymal spermatozoa, oocytes, and embryos were incubated in mediums of variable conditions with and without L-NAME or L-arginine (0.5, 1, 5 and 10 mM) . Fertilization rate and early embryonic development were significantly inhibited by treating sperms or oocytes with L-NAME (93 . 8% vs 66.3%, 92.1% vs 60.3%), but not with L-arginine . In contrast, fertilization rate and early embryonic development were conspicuously reduced when L-NAME or L-arginine was added to the culture media for embryos . Early embryonic development was inhibited by microinjection of L-NAME into the fertilized embryos in a dose-dependent manner, but only by high concentrations of L-arginine . These results suggest that a moderate amount of NO production is essential for fertilization and early embryo development in mice. J Trace Elem Med Biol, 2003, 17(3), 183 - 92 Effect of increasing selenite concentrations, vitamin E supplementation and different fetal calf serum content on GPx1 activity in primary cultured rabbit hepatocytes; Muller AS et al.; Primary rabbit hepatocytes from 6 week old female New Zealand White rabbits (3.0 x 10(6) viable hepatocytes per treatment) were incubated for 24 h or 48 h with two basic variants of the selenium and vitamin E free DMEM/F12-HAM nutrition medium containing 2.5% or 10% fetal calf serum (FCS) . Selenium and vitamin E concentrations of the media were varied by the addition of 0, 10, 50 and 100 ng Se/mL medium as sodium selenite and 100 microg alpha-tocopheryl acetate/mL . Lactic dehydrogenase (LDH) leakage of the hepatocytes was not influenced by the various selenium concentrations of the media, whereas vitamin E addition significantly inhibited LDH release . The activity of cellular glutathione peroxidase (GPx1) was markedly induced by increasing the selenium supplementation of the culture media . Vitamin E supply further enhanced GPx1 induction . In hepatocytes cultivated at the lower serum concentration (2.5% FCS), increasing the selenite concentration of the media raised GPx1 and reduced the intracellular levels of the reduced tripeptide glutathione (GSH) . No vectored relation between the selenium concentration of the media and the activity of superoxide dismutase (SOD) could be observed . After both incubation periods (24 h and 48 h) SOD activity was significantly higher in the cytosol of hepatocytes grown in media containing 10% FCS as compared to cells incubated at the 2.5% FCS level . Furthermore, SOD activity was reduced by the addition of vitamin E to the media . In conclusion the results indicate an effective metabolism of rabbit hepatocytes for selenite even in amounts as low as nanograms . A general cytoprotective role for vitamin E can be shown by its ability to decrease LDH leakage and by the reduction of SOD activity. J Reprod Dev, 2003 Aug, 49(4), 297 - 305 Possible role of interferon-tau on in vitro development of bovine embryos; Takahashi M et al.; The effect of interferon-tau on in vitro development of bovine embryos was investigated . After in vitro fertilization, embryos developed to the morula stage were cultured for 3 days in TCM-199 or CR1 medium containing BSA or FCS supplemented with or without recombinant IFN-tau produced by a baculovirus expression system . Addition of baculovirus-expressed IFN-tau (100 ng/ml) significantly promoted development to the blastocyst stage in both culture media . Addition of E . coli expressed IFN-tau (2 microg/ml) also significantly promoted the embryonic development . Supplementation of BSA or FCS did not affect the growth-promoting effect of IFN-tau . To determine whether the growth-promoting effect of IFN-tau is related to the interferon type I receptors that bind to type I interferon such as IFN-alpha, embryos were cultured with IFN-alpha . Although IFN-alpha significantly promoted the development, a much higher concentration (25 microg/ml) was required than IFN-tau . A reverse transcription polymerase chain reaction analysis revealed the expression of mRNA encoded type-I IFN receptor subunit from morula to blastocyst stage embryos . The overall results suggest a novel function for IFNs in promoting embryonic development and the effect may be related to type-I IFN receptor expressed in the early stages of preimplantation embryos. Fertil Steril, 2004 Feb, 81(2), 287 - 9 Development of culture media for human assisted reproductive technology; Pool TB; Contemporary culture systems in human assisted reproductive technologies meet the metabolic needs of preimplantation embryos by addressing energetic and amino acid requirements in a stage-specific manner . This approach significantly enhances viability compared with the historical use of simple salt solutions or complex somatic cell media. Di Yi Jun Yi Da Xue Xue Bao, 2004 Feb, 24(2), 139 - 43 Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells; Huang ZH et al.; OBJECTIVE: To study the killing effect of adenovirus-mediated double suicide gene under the regulation of kinase domain-containing receptor (KDR) promoter on human umbilical vein endothelial cells (HUVECs) . METHODS: The sequences of human KDR promoter gene, CD gene and TK gene were amplified by PCR, and the plasmid pKDR-CDglyTK was constructed . A two-step transformation protocol was employed for the construction of a recombinant adenoviral plasmid pAdKDR-CDglyTK that was transfected into 293 packaging cells to further multiply and purify the adenovirus . HUVECs were infected by the resultant recombinant adenovirus of different multiplicities of infection (MOI), and the infection rate was measured by observing the expression of green fluorescence protein (GFP) . The infected cells were cultured in the culture media containing ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the killing effects were evaluated . RESULTS: Recombinant adenovirus AdKDR-CDglyTK were successfully constructed, which could efficiently infect HUVEC cells, with the infection rate associated with the MOI of the recombinant adenovirus . HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of the recombinant adenovirus . The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC . CONCLUSIONS: Prodrug/KDR-CdglyTK system is effective in killing HUVEC cells, and its killing effect is correlated with the concentration of the prodrugs and the MOI of the recombinant adenovirus . Combination of the two prodrugs produces stronger killing effect on the cells. Genet Mol Res, 2002 Jun 30, 1(2), 128 - 30 A technique to obtain fibroblast cells from skin biopsies of living bats (Chiroptera) for cytogenetic studies; Moratelli R et al.; We developed a procedure to obtain fibroblasts from bat skin . A small fragment of the ear is removed under ether anesthesia . This material is then cut up into small pieces and cultured in standard cell culture media . Very good quality chromosome preparations for cytogenetic studies are obtained in about three weeks . Secondary cultures can be used for other biological studies . This procedure does not require sacrificing the animals. Cancer Res, 2004 Feb 1, 64(3), 1037 - 43 Neutrophil-derived TNF-related apoptosis-inducing ligand (TRAIL): a novel mechanism of antitumor effect by neutrophils; Koga Y et al.; To detect the novel genes expressed uniquely in neutrophils and elucidate their function, the gene expression pattern was compared by using cDNA microarray containing 240 cytokine genes between the neutrophils and peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors . Twenty-six genes, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were expressed in neutrophils at a level >10 times higher than that seen in phytohemagglutinin-stimulated PBMCs . The amounts of mRNA and protein of TRAIL were quantified by real-time reverse transcription-PCR and ELISA, respectively . TRAIL was expressed in resting neutrophils at the mRNA and protein levels, and its expression was enhanced after stimulation with IFN-gamma . Neutrophils expressed TRAIL on the cell surface and released it into the culture media . The cytotoxicity of neutrophil-derived TRAIL against Jurkat cells was determined by flow cytometry using FITC-conjugated annexin V . When Jurkat cells were cultured with neutrophils in the presence of IFN-gamma, the number of Jurkat cells undergoing apoptosis increased, and such increase depended on the effector:target ratio . This cytotoxicity was suppressed partially by adding anti-TRAIL antibody to the media . Neutrophils may exert their own antitumor effect by TRAIL . A microarray analysis was found to be a useful tool for detecting novel genes that are suggested to play unknown roles in the neutrophil function. Biotechnol Appl Biochem, 2004 Oct, 40(Pt 2), 201 - 7 Characterization and properties of acid phosphatases with phytase activity produced by Aspergillus caespitosus; Guimaraes LH et al.; High levels of thermostable acid phosphatases were produced by Aspergillus caespitosus in culture media supplemented with xylan birchwood or agricultural residues, as carbon sources . The optimal culture conditions for production of phosphatases were 40 degrees C and pH 6.0 . Extra- and intra-cellular acid phosphatases were purified by chromatography on DEAE-cellulose, followed by concanavalin A-Sepharose affinity separation . Both extra- and intra-cellular enzymes were glycoproteins showing 63.0 and 58.3% of carbohydrate content respectively . Molecular masses estimated on Sepharose CL-6B column were 186 and 190+/-15 kDa, and 84 and 74+/-5 kDa according to SDS/PAGE, for extra- and intra-cellular acid phosphatases respectively . Taken together, these results suggest that both native enzymes were homodimers . Optimum temperature and pH for both phosphatase activities were 80 degrees C and 5.5 respectively . The extra- and intra-cellular acid phosphatases were stable for more than 60 min at 60 degrees C . The extracellular acid phosphatase was slightly inhibited by NaF, in contrast with the significant inhibition of the intracellular form . KH(2)PO(4) inhibited both activities equally . Both extra- and intracellular acid phosphatases were tartarate-resistant . Among several phosphorylated substrates used, the extracellular enzyme preferentially hydrolysed p-nitrophenyl phosphate . Kinetic parameters calculated for the hydrolysis of p-nitrophenyl phosphate by extracellular acid phosphatase were h (Hill coefficient)=1.2, K(0.5)=0.082 mM and V(max)=4.43 units/mg, whereas the intracellular enzyme exhibited Michaelian kinetics with K(m)=0.029 mM and V(max)=0.082 unit/mg . Phytase activity was also observed for both the enzymes, suggesting that they could be useful for biotechnological applications. Lupus, 2004, 13(1), 45 - 53 Neuroendocrine dopaminergic regulation of prolactin release in systemic lupus erythematosus: a possible role of lymphocyte-derived prolactin; Mendez I et al.; Prolactin (PRL) secretion by the pituitary is under the control of dopamine . Hyperprolactinemia has been found in patients with systemic lupus erythematosus (SLE) and seems to be associated with clinical activity . T-lymphocytes express PRL and those from SLE patients appear to secrete more PRL than controls . In this study, immuno-(RIA) and bio-(BIO) assayable PRL in both serum and culture media of peripheral blood mononuclear cells (PBMNC) from SLE and control subjects were evaluated in the basal state and in response to 10 mg oral administration of metoclopramide, a dopamine receptor antagonist . Prolactin size heterogeneity in serum and culture media and PRL gene transcription in PBMNC were also studied . Basal serum RIA-PRL, BIO-PRL and the BIO/RIA ratio were similar in both groups . The serum BIO-PRL response after metoclopramide was higher than RIA-PRL in SLE, and this increment was also greater than in control subjects . PBMNC from SLE subjects secreted and produced more BIO-PRL . After metoclopramide, secretion and production of PRL increased only in PBMNC from control women and not in those from SLE patients . Our results demonstrated an increased central dopaminergic tone in SLE and suggest that lymphocyte-derived PRL might contribute to alter the functional activity of the hypothalamic dopaminergic system in SLE attempting to maintain serum PRL within a physiological range. Lupus, 2004, 13(1), 17 - 23 Intrauterine fetal death in mice caused by cytomegalovirus-derived peptide induced aPL antibodies; Gharavi AE et al.; Immunization of mice with beta2 glycoprotein I (beta2GPI) and also with GDKV, a synthetic peptide representing the phospholipid (PL)-binding site of beta2GPI, induced pathogenic aPL antibodies that bind and activate endothelial cells, enhanced thrombus formation and caused fetal death in pregnant mice . TIFI is a PL-binding peptide spanning the Thr101-Thr120 of ulb0-hcmva from human cytomegalovirus (CMV), which shares structural similarity with the PL-binding site of beta2GPI . Immunization with this peptide induced pathogenic aPL and anti-beta2GPI antibodies in mice . These antibodies activated endothelial cells and enhanced thrombus formation in vivo, but whether these antibodies cause fetal death in mice is not known . The objective of this study was to examine the effects of these antibodies on pregnancy outcome in mice . Two groups of pregnant BALB/c mice were injected with either hybridoma supernatant containing D3/AC10, a CMV-peptide-induced monoclonal aPL, at days four, eight and 12 of the pregnancy, 100 microg per mouse (study group) or with culture media alone (control group) . The litter size was significantly smaller in the study group (4.80 +/- 1.15 versus 7.28 +/- 0.18, t = - 2.526, P < 0.03) . In conclusion, aPL induced by CMV peptides may have pathogenic properties similar to human autoimmune aPL . These findings further support the hypothesis that at least