Exp Eye Res, 2003 Aug, 77(2), 195 - 202 Effects of baicalin, baicalein, and wogonin on interleukin-6 and interleukin-8 expression, and nuclear factor-kappab binding activities induced by interleukin-1beta in human retinal pigment epithelial cell line; Nakamura N et al.; The objective of the study was to investigate the effects of baicalin, baicalein, and wogonin (plant flavonoids) on interleukin-6 (IL-6) and interleukin-8 (IL-8) protein production, mRNA expression, and nuclear factor-kappaB (NF-kappaB) binding activities induced by interleukin-1beta (IL-1beta) in human retinal pigment epithelial cell line (ARPE-19) cells . To induce IL-6 and IL-8 mRNA expression and protein levels, IL-1beta was added to serum-free medium of ARPE-19 cells and incubated . The flavonoids were added to the medium . IL-6 and IL-8 in the media were measured using enzyme-linked immunosorbent assay . Both IL-6 and IL-8 mRNA were measured by semiquantitative reverse transcription polymerase chain reaction . The binding activities of the transcription factor NF-kappaB complexes to IL-6 and IL-8 were measured by electrophoretic mobility shift assay . IL-6 and IL-8 in the culture media of ARPE-19 cells were increased by IL-1beta in a dose-dependent manner . Baicalin did not suppress IL-1beta-induced IL-6 and IL-8 production, but dexamethasone, baicalein, and wogonin, significantly suppressed IL-6 and IL-8 production . Elevation of IL-6 and IL-8 mRNA was not suppressed by baicalin but was significantly suppressed by dexamethasone, baicalein, and wogonin . NF-kappaB binding activities were not suppressed by baicalin and baicalein, but was suppressed by wogonin . Wogonin and baicalein inhibited IL-1beta-induced IL-6 and IL-8 mRNA and protein production in ARPE-19 cells . The data suggest that wogonin may inhibit IL-6 and IL-8 mRNA expression via the suppression of NF-kappaB binding activities. Genesis, 2003 Jul, 36(3), 129 - 33 Two-phase chemically defined culture system for preimplantation rat embryos; Zhou Y et al.; A limiting factor in the development of new technologies and transport of rats worldwide has been the inability to robustly culture preimplantation embryos . Previously, culture in vitro to the blastocyst stage from one-cell embryos was successful only if the one-cell embryos were isolated near the time of the first cleavage and from only a few strains . Here we report the use of commonly available, chemically defined culture media to overcome these limitations . In vitro culture of young one-cell embryos using common embryo media (KSOM, BMOC, or HTF) for 18-22 h followed by culture in mR1ECM medium allows the successful in vitro development of blastocysts from one-cell embryos after 5 days from both outbred (SD) and inbred strains of rat (WF, LEW, F344, and PVG) . This system allows the parthenogenetic development of chemically activated, unfertilized oocytes to the blastocyst stage . Embryos cultured in this system develop to term and are live-born following transfer to surrogate mothers . Cancer Gene Ther, 2003 Aug, 10(8), 571 - 82 Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells . Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy; Schwartz PS et al.; Cytochrome P450-based gene therapy can substantially increase the sensitivity of tumor cells to P450-activated cancer chemotherapeutic prodrugs such as cyclophosphamide (CPA) without increasing host toxicity . While the role of 4-OH-CPA, the primary active metabolite of CPA, in eliciting tumor cell death is well established, the effect of 4-OH-CPA exposure on the capacity of P450-expressing tumor cells for continued metabolism and activation of CPA has not been investigated . The present study addresses this question and characterizes the impact of CPA dose and treatment schedule on the ability of P450-expressing tumor cells to sustain prodrug activation over time . 9L gliosarcoma cells expressing human P450 2B6 and treated with CPA in a continuous manner exhibited a time- and CPA dose-dependent decrease in P450-catalyzed CPA 4-hydroxylase activity . This decrease reflects a selective, 4-OH-CPA-induced loss of cellular P450 protein content . By contrast, when the P450-expressing tumor cells were treated with CPA as a single 8 hours exposure, cellular CPA 4-hydroxylase activity and P450 protein expression were substantially prolonged when compared to continuous prodrug treatment . This schedule-dependent effect of CPA was influenced by the level of P450 protein expressed in the tumor cells . At high P450 protein and activity levels, which could be achieved by culturing the tumor cells at high cell density, net production and release of 4-OH-CPA into the culture media was increased substantially . This increase fully offset the decline in CPA 4-hydroxylase activity as the tumor cells underwent CPA-induced apoptotic death . These findings demonstrate the impact of CPA dose and treatment schedule on the efficacy of P450 gene-directed enzyme prodrug therapy, with bolus CPA treatment being compatible with sustained expression of P450 protein and maintenance of P450-dependent prodrug activation by the target tumor tissue. Mycoses, 2003 Apr, 46(3-4), 157 - 8 Keratomycosis due to Trichophyton mentagrophytes; Shenoy R et al.; We report the case of an Omani lady who presented with keratitis that grew Trichophyton mentagrophytes on culture media and responded to treatment with topical fluconazole. Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9512 - 7 Epub 2003 Jul 16. Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor; Kiaris H et al.; Existing evidence indicates that, in addition to its neuroendocrine action, growth hormone-releasing hormone (GHRH) acts directly on several nonpituitary tissues, especially neoplasms, and stimulates cell proliferation . We have recently reported that a splice variant of the receptor (SV1) is expressed in various normal tissues and particularly in tumor tissues, producing mitogenic effects on GHRH binding . By using HEC-1A human endometrial carcinoma cells, which express endogenous SV1, we show that, in addition to its ability to mediate the mitogenic effects of GHRH, SV1 also possesses relatively high intrinsic, ligand-independent activity . By using an antisense RNA-based approach we found that SV1 ablation reduces the efficacy of colony formation and the rate of cell proliferation of HEC-1A cells in the absence of exogenous GHRH, and decreases their sensitivity to GHRH when the neurohormone is added to the culture media . This ligand-independent stimulation of cell proliferation appears to be a characteristic property of the truncated form of the receptor, because the expression of SV1 and not of the full-length GHRH receptor stimulated the proliferation of 3T3 fibroblasts in the absence of exogenous GHRH, whereas both forms mediated the proliferative effects of GHRH . Evaluation of 21 specimens of human primary endometrial carcinoma for expression of SV1 by immunohistochemistry indicated that in contrast to the GHRH receptor, which is absent, SV1 is expressed in approximately 43% of the specimens . These findings indicate that SV1 can operate in a ligand-independent as well as a ligand-dependent manner . The overexpression of this form of GHRH receptor may be associated with carcinogenesis. J Med Microbiol, 2003 Aug, 52(Pt 8), 675 - 9 Ultrastructural observation of Helicobacter pylori in glucose-supplemented culture media; Sato F et al.; Helicobacter pylori in the human gut can be divided morphologically into spiral and coccoid forms . The spiral form is known to change into the coccoid form in culture in vitro . The ultrastructural changes and culturability of H . pylori were studied in medium supplemented with different concentrations of glucose . H . pylori ATCC 43504(T) was cultured in liquid medium containing 10 % heat-inactivated horse serum supplemented with glucose (at 0, 10, 100, 300 and 500 mM) for 7 days . Bacterial ultrastructure and culturability were examined daily . With extended time in culture, the spiral forms had transformed into coccoid forms in all media . The coccoid forms could be further divided into two types, A and B, by electron microscopy . The type A coccoid form had an irregular surface with few flagella and indistinct cytoplasmic membrane . The type B coccoid form had a better-maintained integral membrane structure and was the dominant form in 300 mM glucose-supplemented medium . The highest culturability was obtained using 300 mM glucose-supplemented medium . Based on observations of ultrastructural changes in relation to the culturability data, the coccoid forms could be categorized into three stages: dying, viable but non-culturable and proliferating organisms . The optimal glucose concentration for H . pylori culture in this liquid medium culture experiment was approximately 300 mM. Spine, 2003 Jul 15, 28(14), 1528 - 33 Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs: an ex vivo study; Ariga K et al.; STUDY DESIGN: Various amounts of static mechanical load were applied to mouse intervertebral discs in organ cultures . The apoptosis then was examined using nick end labeling . Two mitogen-activated protein kinase (MAPK) inhibitors were added to the medium . OBJECTIVES: To establish an experimental model for detecting factors regulating chondrocyte apoptosis induced by mechanical stress, and to determine the role of MAPK and p38 in the stress-induced apoptotic pathway of endplate chondrocytes . SUMMARY OF BACKGROUND DATA: The cause of degenerative change in the cartilaginous endplate (CEP) remains unclear . The authors' previous findings using a mouse model suggested that apoptosis in the cartilaginous endplate may play a role in intervertebral disc degeneration, and that mechanical stress may induce apoptosis . If apoptosis of endplate chondrocytes is involved in the cascade of intervertebral disc degeneration, then how apoptosis is induced by mechanical stress should be important in preventing disc degeneration . However, the mechanism of apoptosis induced by mechanical stress remains unclear . METHODS: Mouse coccygeal discs were harvested and organ cultured . Various static compression loads (0, 0.2, 0.4, 0.8, and 1.0 MPa) were applied on intervertebral discs placed in culture bottles for 24 hours . Paraffin-embedded sections of the harvested discs were stained using Safranin-O and the nick end labeling procedure . The apoptotic cells were counted in the cartilaginous endplate and junctional anulus fibrosus of each intervertebral disc . In addition, U0126 (MAPK inhibitor) and SB202190 (p38 inhibitor) were added to the culture medium to determine their regulatory roles in the apoptosis of endplate chondrocytes induced by mechanical load . RESULTS: Histologically, loaded discs became bulged, and the disc space became narrow . Apoptosis was absent in discs without load, but was particularly noticeable in loaded discs (load weight, 1.0 MPa) . The number of apoptotic cells increased depending on the weight of the load . The two MAPK inhibitors significantly increased the number of apoptotic cells . CONCLUSIONS: Chondrocyte apoptosis was induced using a static mechanical load especially in the cartilaginous endplate in an organ culture . Apoptosis occurred similarly to previous findings using an in vivo model . This culture system thus reflected the apoptosis demonstrated in vivo . Because biologically active reagents such as MAPK inhibitors can be simply added to culture media, this system may be a useful method for detecting factors that influence apoptosis induced by mechanical stress . Both MAPK inhibitors increased the occurrence of apoptosis . This suggests that these two MAPKs can counteract the apoptotic pathway induced by mechanical stress. Prostate, 2003 Sep 1, 56(4), 239 - 49 Human prostate cancer cells and xenografts are targeted and destroyed through luteinizing hormone releasing hormone receptors; Leuschner C et al.; BACKGROUND: A conjugate of a lytic peptide, hecate, and a 15-amino acid segment of the beta-chain of chorionic gonadotropin (CG) destroyed human prostate xenografts in nude mice by targeting LH receptors . Since these xenografts also express LHRH receptors, we prepared a LHRH-hecate conjugate and tested its ability to destroy PC-3 cells in vitro and in vivo . MATERIALS AND METHODS: LHRH-hecate was added to cultures of PC-3, BRF 41 T, DU145, and LNCaP cells in the presence and absence of steroids . PC-3 xenografts were established in nude male mice, which were treated with LHRH-hecate . RESULTS: Injections of LHRH-hecate resulted in tumor growth arrest and marked reduction of tumor burden (62.2 mg/g body weight in saline controls vs . 10.5 mg/g body weight in treated mice; P < 0.0001); unconjugated LHRH and hecate had no effect on tumor burden and tumor viability (48.5 mg/g body weight in LHRH treated animals vs . 63.2 mg/g body weight in hecate treated mice) . Marked tumor necrosis occurred in conjugate treated mice . Removal of steroids from the culture media decreased the sensitivity of LNCaP and PC-3 cells to the LHRH-hecate; adding estrogen restored the sensitivity . CONCLUSIONS: LHRH-hecate may be effective in treating hormone dependent and independent prostate cancers . J Cosmet Sci, 2003 May-Jun, 54(3), 229 - 38 Inhibition of matrix metalloproteinase-1 and -2 expression using nitric oxide synthase inhibitors in UV-irradiated human dermal fibroblasts; Choe T et al.; The production of matrix metalloproteinases (MMP) by UV-irradiated skin fibroblasts and the degradation of the extracellular matrix by these enzymes is known as one of the main causes of photoaging . Recently, the Fisher group showed that MMP expression is mainly regulated by members of the mitogen-activated protein kinase family such as extracellular signal-regulated kinase, c-Jun amino-terminal kinase, and p38, each of which forms a signaling pathway . In this work, we initially examined the effect of nitric oxide (NO) and nitric oxide synthase (NOS) inhibitors on the production of MMP-1 and MMP-2 by human dermal fibroblasts (HDF) . NO is a multifunctional messenger molecule generated from L-arginine and can activate guanylate cyclase to increase cGMP . We found that treatment of HDF with an NO donor, sodium nitroprusside (50 microM), enhanced the expression of MMP-1 and -2 by 153% and 243%, respectively, and treatment by 8-Br-cGMP enhanced MMP-1 and -2 expression by 137% and 254%, respectively . When UV-irradiated HDF was treated with NOS inhibitors such as aminoguanidine (AG) and baicalein (BAC), there resulted a decrease in MMP production . When 20 microM of BAC was added in the culture media of UV-irradiated HDF, only 40% of MMP-1 and 42% of MMP-2 was produced, compared to the case without BAC . Taken together, we concluded that the production of MMP-1 and -2 by UV-irradiated HDF is regulated through the signaling pathway involving NO and that it can be downregulated using NOS inhibitors. Exp Parasitol, 2002 Nov-Dec, 102(3-4), 150 - 6 Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases; de Melo AC et al.; The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE . Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin . Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate . All C . guilhermei proteinases were incapable of using bovine serum albumin . C . guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source . The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles. Brain Res Dev Brain Res, 2003 Jul 12, 143(2), 207 - 16 Aggrecan regulates telencephalic neuronal aggregation in culture; Domowicz MS et al.; Proteoglycans have been suggested to play roles in pattern formation in the developing central nervous system . In the chick embryo, aggrecan, a chondroitin sulfate proteoglycan, has a regionally-specific and developmentally-regulated expression profile . Telencephalic neuronal cultures, when aggregated, exhibit aggrecan expression patterns comparable to those observed in vivo . The chicken mutation nanomelia produces a truncated aggrecan species that cannot be processed further and is not secreted . Neurons from normal and nanomelic chick embryo telencephalon were scored for aggregate formation and analyzed for distribution of aggrecan protein and expression of aggrecan mRNA . Distinctly different pattern formation, with respect to aggregate size (smaller) and number (fewer) were observed in poly-L-lysine plated neuronal cultures derived from nanomelic embryos when compared to those derived from normal embryos . Significantly, the nanomelic phenotype was subsequently rescued upon addition of the brain-specific form of aggrecan . Modulation of neuronal aggregate formation was mimicked by treatment with chondroitinase ABC but not other glycanases, and was rescued by addition of chondroitin 6-sulfate to the culture media . Lastly, although broad and diffuse distribution of aggrecan among the cell aggregates in the culture paradigm was observed by immunocytochemistry, mRNA in situ hybridization revealed that only a small population of cells in the center of the aggregates was responsible for the production of the secreted aggrecan found associated with neuronal aggregates . These studies suggest a function for aggrecan as a diffusible signal in CNS histomorphogenesis. J Neurosci, 2003 Jul 9, 23(14), 6030 - 40 Retinoschisin, a photoreceptor-secreted protein, and its interaction with bipolar and muller cells; Reid SN et al.; Usually, photoreceptors interact with other retinal cells through the neurotransmitter glutamate . Here we describe a nonsynaptic interaction via a secreted protein, retinoschisin . Using in situ hybridization, we found that from early postnatal life retinoschisin mRNA is present only in the outer retina of the mouse, and with single-cell RT-PCR we demonstrated its localization in rod and cone photoreceptor cells but not in Muller cells . Western blot analyses of proteins from cultured ocular tissues and microdissected outer and inner retinas, as well as from the culture media of these samples, showed that retinoschisin is secreted from the photoreceptor cells . Immunostaining of permeabilized and nonpermeabilized dissociated retinal cells revealed that retinoschisin is mainly inside and outside the photoreceptors, outside bipolar cells, and associated with plasma membranes of Muller cells and inside their distal processes . Because we showed previously that retinoschisin is distributed all over the retina, our current data suggest that after synthesis and secretion by the photoreceptors, retinoschisin reaches the surface of retinal cells and mediates interactions/adhesion between photoreceptor, bipolar, and Muller cells, contributing to the maintenance of the cytoarchitectural integrity of the retina . These interactions may not occur when the gene encoding retinoschisin is mutated, as it occurs in X-linked juvenile retinoschisis, a disease that results in morphological and electrophysiological defects of the retina. Am J Reprod Immunol, 2003 Apr, 49(4), 230 - 8 Feasibility to generate monocyte-derived dendritic cell from coculture with melanoma tumor cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4; Kim YT et al.; OBJECTIVE: We investigated how melanoma cells and membrane-bound granulocyte/macrophage colony stimulating factor (mbGM-CSF) melanoma cell lines affect the differentiation of dendritic cells (DC) from CD14+ monocytes . METHOD OF STUDY: The malignant melanoma cell lines (Conley and Jorp) and mbGM-CSF-positive cell lines (Conley B-F8 and Jorp C-E6) were cultured and cell-free supernatants were collected from each cell line cultures to assess the GM-CSF level . Adherent monocytes were cocultured for 6-7 days with irradiated mbGM-CSF and wild type melanoma cells (50 Gy) at each 1:1 and 0.1:1 ratio in six-well culture plates in ex vivo culture medium containing interleukin (IL)-4 . Immunophenotyping was performed by triple color immunofluorescence staining with flow cytometry analysis . RESULTS: GM-CSF was detected at low levels in the culture supernatants of Conley B-F8 (0.48 ng/10(6) cells/24 hr), whereas there was no detectable GM-CSF in that of Conley melanoma cell line . Monocytes cultured with GM-CSF/IL-4 generated the expression of high levels of HLA DR, CD1a and CD86, while the expression of CD14 and CD83 were in low amounts . However, the addition of GM-CSF to these cultures resulted in an increased expression of these markers and decreased that of CD14 . Monocytes cocultured with Jorp C-E6 illustrated similar expression pattern of CD1a, HLA DR and CD14 in the presence or absence of GM-CSF as Conley B-F8 melanoma cell line . Monocytes cocultured with Conley B-F8 melanoma cells at 1:1 and 0.1:1 ratio showed no significant difference in expression of CD1a, CD14 and CD83 between the two ratios . CONCLUSION: Our results illustrate the feasibility to generate monocyte-derived DC from coculture with melanoma tumor cells in the presence of GM-CSF and IL-4 . However, mbGM-CSF tumor cells did not significantly enhance the DC differentiation through juxtacrine stimulation unless soluble GM-CSF was added in the culture media. Gastroenterology, 2003 Jul, 125(1), 117 - 25 Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II; Bataller R et al.; BACKGROUND & AIMS: The renin-angiotensin system plays an important role in hepatic fibrogenesis . In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis . It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II . The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II . METHODS: Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells) . Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells . Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction . Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry . RESULTS: Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II . In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media . Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells . Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect . Vasodilator substances markedly attenuate the effect of endothelin-1 . CONCLUSIONS: After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II . These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver. Cytotherapy, 2003, 5(3), 259 - 72 Ex vivo expansion of the highly cytotoxic human natural killer-92 cell-line under current good manufacturing practice conditions for clinical adoptive cellular immunotherapy; Tam YK et al.; BACKGROUND: Adoptive transfer of ex vivo expanded cytotoxic immune cells has become a viable strategy for treatment of malignant disease . Natural killer (NK)-92, a highly cytotoxic, IL2-dependent human NK cell-line, is an excellent candidate as an immunotherapeutic agent, being active for prolonged periods following irradiation and IL2 deprivation, non-toxic and non-immunogenic, and easily expanded . A number of clinical trials using NK-92 for different indications are currently underway . The aim of this study was to develop current good manufacturing practice (cGMP)-compliant expansion methodology for NK-92 . METHODS: The ability to expand NK-92 ex vivo was evaluated . Serum-free culture media, as well as media supplements (IL2, serum/plasma/albumin), culture containers and feeding regimens were compared for their ability to support expansion, viability and cytotoxicity of NK-92 cells . RESULTS: NK-92 cells can be expanded in X-Vivo 10 serum-free media with 500 U/mL of rhIL2 (Proleukin), and 2.5% human serum/plasma to achieve concentrations sufficient to treat patients with >5210(10) cells . The protocol involves cultures initiated at 2.5210(5) cells/mL in 25 mL in 1 L Vuelife culture bags, with addition of fresh media plus IL2 every 3 days to maintain an optimal density of NK-92 cells for expansion . Daily disruption of cell aggregates enhances NK-92 cells expansion and viability during the culture period . Final yields of approximately 1.1-1.3210(6) cells/mL in a 1.2 L volume (1.36-1.56210(9) cells; 218-250 fold expansion) over 15-17 days is achievable under cGMP-compliant conditions with >85% viability . The feasibility of this approach has been shown in ongoing clinical trial with NK-92 . DISCUSSION: We describe a protocol that allows for >200-fold expansion of NK-92 cells within a 2-2.5 week period under GMP standards, in quality and quantity suitable for clinical adoptive immunotherapy. Life Sci, 2003 Jul 25, 73(10), 1299 - 313 Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors; Kojima-Yuasa A et al.; Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer . However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established . In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/Kinase insert domain containing receptor (Flk-1/KDR) in vitro using human umbilical vein endothelial cells (HUVECs) . GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase . The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods . These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors. Biochem Biophys Res Commun, 2003 Jul 18, 307(1), 157 - 64 Antioxidant inhibits tamoxifen-DNA adducts in endometrial explant culture; Sharma M et al.; Fresh human endometrial explants were incubated for 24h at 37 degrees C with either tamoxifen (10-100 micro M) or the vehicle (0.1% ethanol) . Three metabolites namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in the culture media . Tissue size was limited but DNA adducts formed by the alpha-hydroxytamoxifen pathway were detected using authentic alpha-(deoxyguanosyl-N(2)) tamoxifen standards . Relative DNA-adduct levels of 2.45, 1.12, and 0.44 per 10(6) nucleotides were detected following incubations with 100, 25, and 10 micro M tamoxifen, respectively . The concurrent exposure of the explants to 100 micro M tamoxifen with 1mM ascorbic acid reduced the level of alpha-hydroxytamoxifen substantially (68.9%) . The formation of tamoxifen-DNA adducts detectable in the explants from the same specimens exposed to 100 micro M tamoxifen with 1mM ascorbic acid were also inhibited . These results support the role of oxidative biotransformation of tamoxifen in the subsequent formation of DNA adducts in this tissue. Biochem Biophys Res Commun, 2003 Jul 18, 307(1), 69 - 73 Amphiphilic properties of (-)-epicatechin and their significance for protection of cells against peroxynitrite; Schroeder P et al.; The dietary flavanol (-)-epicatechin protects against nitration and oxidation reactions of the inflammatory mediator peroxynitrite in hydrophilic and hydrophobic environments . Bioavailability and cellular uptake of (-)-epicatechin are not yet fully characterized . Here, the octanol/buffer partition coefficient of (-)-epicatechin is observed to be 1.5, indicating that the flavanol is soluble in aqueous as well as lipophilic cellular phases, thus capable of permeating the cell membrane . In line with this, the ability of murine aortic endothelial cells (MAECs) to remove (-)-epicatechin from cell culture media is demonstrated . Epicatechin accumulates in cells, likely due to epicatechin binding to cellular proteins . Even after repeated washing, (-)-epicatechin accumulated by MAEC affords protection of the cells against peroxynitrite-induced nitration of protein tyrosyl residues and against oxidation of intracellular dichlorodihydrofluorescein. Mol Plant Microbe Interact, 2003 Jul, 16(7), 567 - 79 Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions; Lopez-Lara IM et al.; Membrane lipids in most bacteria generally consist of the glycerophospholipids phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE) . A subset of bacteria also possesses the methylated derivatives of PE, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine (PC) . In Sinorhizobium meliloti, which can form a nitrogen-fixing root nodule symbiosis with Medicago spp., PC can be formed by two entirely different biosynthetic pathways, either the PE methylation pathway or the recently discovered PC synthase pathway . In the latter pathway, one of the building blocks for PC formation, choline, is obtained from the eukaryotic host . Under phosphorus-limiting conditions of growth, S . meliloti replaces its membrane phospholipids by membrane-forming lipids that do not contain phosphorus; namely, the sulfolipid sulfoquinovosyl diacylglycerol, ornithine-derived lipids, and diacylglyceryl-N,N,N-trimethylhomoserine . Although none of these phosphorus-free lipids is essential for growth in culture media rich in phosphorus or for the symbiotic interaction with the legume host, they are expected to have major roles under free-living conditions in environments poor in accessible phosphorus . In contrast, sinorhizobial mutants deficient in PC show severe growth defects and are completely unable to form nodules on their host plants . Even bradyrhizobial mutants with reduced PC biosynthesis can form only root nodules displaying reduced rates of nitrogen fixation . Therefore, in the cases of these microsymbionts, the ability to form sufficient bacterial PC is crucial for a successful interplay with their host plants. Am J Physiol Cell Physiol, 2003 Aug, 285(2), C425 - 32 Increase in epidermal growth factor receptor protein induced in osteoblastic cells after exposure to flow of culture media; Ogata T; To investigate how bone cells respond to mechanical stimuli, we subjected osteoblastic cells to fluid flow . We and others already reported that in a culture system of osteoblast-like cells, ERK1/2, Shc, and other proteins were tyrosine-phosphorylated by medium flow and the early response gene, egr-1 or c-fos mRNA, increased . These are the same as events found after stimulation by various growth factors . Moreover, because there were also reports suggesting that growth factor signaling is involved in the responses to mechanical stimuli, we examined the change in epidermal growth factor (EGF) receptor in the cells exposed to medium flow . The results demonstrated that EGF receptor protein increased after exposure to medium flow . This increase did not occur without serum in media, and the addition of EGF restored it . Furthermore, leupeptin blocked this increase . These results suggest that degradation of EGF-occupied EGF receptor by leupeptin-sensitive protease(s) in endosomes decreased with exposure to medium flow . This was presumed to participate, at least in part, in signaling of fluid flow. Hua Xi Kou Qiang Yi Xue Za Zhi, 2003 Apr 20, 21(2), 92 - 4 {Experimental study on the chondrogenesis potentiality of marrow stromal cell under the induction of transforming growth factor-beta}; Chen F et al.; OBJECTIVE: Seed cell study is an essential area in the research of tissue engineering . To evaluate the potentiality of marrow stromal cell(MSCs) as seed cell in the regeneration of tissue engineered cartilage, formation of cartilage nodules by culture expanded MSCs pellets under the induction of TGF-beta was investigated . METHODS: MSCs were cultured and expanded in vitro . Cell pellets containing 1 x 10(6) MSCs were obtained by centrifuging MSCs solution at 1,000 r/min in 5 ml centrifugation tube . Pellets were exposed to cell culture media containing 20 ng/ml TGF-beta for 7 days and then cultured for another 7 and 21 days . The nodules were moved out of the tube and cartilage formation was observed by stereomicroscope, light microscope and electronic microscope . RESULTS: 10 days after exposure to TGF-beta, pellets contracted and formed small and round nodules on the bottom of the tubes . The nodules grew bigger slowly and reached maximal diameter of 1.8 mm in 28 days . The surface of the nodules was smooth and bright white . Histological examination showed that extra cellular matrix formed in 14 days and in some areas cells situated in lacuna . In 28 days' specimens, a lot of cells situated in lacuna could be observed and the histological appearance looked much similar to cartilage . Electronic microscope observation demonstrated that in 28 days' specimens a large amount of collagen fiber existed . CONCLUSION: Under the induction of TGF-beta, MSCs could differentiate into chondrogenesis cell and form cartilaginous nodules in vitro . This indicated that MSCs could be the potential seed cells in the regeneration of cartilage employing method of tissue engineering. Mikrobiyol Bul, 2002 Jul-Oct, 36(3-4), 229 - 35 {Comparison of BBL mycobacteria growth indicator tube (MGIT) method, BACTEC radiometric system and Lowenstein-Jensen culture media for the detection of mycobacteria in clinical specimens}; Alp A et al.; The need for more rapid diagnostic tests for early detection of mycobacteria in clinical specimens has become more apparent for the control of tuberculosis . Several commercial systems, as well as conventional media, are available for the culture of acid-fast bacteria (AFB) . The aim of this study was to evaluate the Mycobacteria Growth Incubator Tube (MGIT), BACTEC 460 and Lowenstein-Jensen (LJ) media for the recovery of mycobacteria from processed clinical specimens . A total of 75 clinical specimens which were found positive for AFB in smear microscopy were included to the study, and of them 73 were grown in MGIT system, 70 in BACTEC 460 system and 74 on the LJ slants . Average periods for the detection of mycobacteria for BACTEC 460, MGIT systems and LJ medium were 7.3 days, 10.9 days and 19.4 days, respectively . As a result, it was found that the recovery of mycobacteria in MGIT system is comparable to BACTEC 460 system . It appears to be a safer system than BACTEC 460 system, and may be used for detection of mycobacteria in laboratories desiring a non-radioactive method. Laryngoscope, 2003 Jul, 113(7), 1113 - 7 Volume analysis of preadipocyte injection for vocal cord medialization; Glatz FR et al.; OBJECTIVE: To compare the volume retention of injected preadipocytes with that of standard fat injection in a paralyzed rabbit true vocal cord . STUDY DESIGN: Prospective analysis with blinded data collection . METHODS: Thirteen New Zealand white rabbits were divided into two groups . Group 1 consisted of seven animals undergoing left-side vocal cord paralysis by resection of a 1-cm segment of the left-side recurrent laryngeal nerve and abdominal fat harvest for isolation of preadipocytes . Preadipocytes were cultured under sterile conditions in cell culture media . Animals in group 2 also underwent left-side vocal cord paralysis without fat harvest . After 10 to 14 days, in a second procedure, group 1 underwent injection of 0.1 mL cultured autologous preadipocytes, and group 2 underwent routine injection of 0.1 mL abdominal fat harvested during the same procedure . At 6 and 12 months, volumetric analysis was performed . RESULTS: Volume analysis at 6 months showed a mean volume of 0.029 mL retained fat in group 2 representing a retention of approximately 29% (SD = 0.023) of the original injected volume . Retention in group 1 animals approximated 0.002 mL (SD = 0.0024) or 2% of the injected volume . Analysis at 12 months showed a mean volume of 0.008 mL (SD = 0.0078) in group 2 and of 0.002 mL (SD = 0.0015) in group 1 . Group 2 showed significantly higher volumes of the injected fat at 6 and 12 months (P <.033) . CONCLUSION: Volumes obtained with standard fat injection were superior to those obtained with preadipocyte injection at both 6 and 12 months. J Neurosci Res, 2003 Jul 15, 73(2), 260 - 9 Pyruvate protection against beta-amyloid-induced neuronal death: role of mitochondrial redox state; Alvarez G et al.; The mechanism by which beta-amyloid protein (A beta) causes degeneration in cultured neurons is not completely understood, but several lines of evidence suggest that A beta-mediated neuronal death is associated with an enhanced production of reactive oxygen species (ROS) and oxidative damage . In the present study, we address whether supplementation of glucose-containing culture media with energy substrates, pyruvate plus malate (P/M), protects rat primary neurons from A beta-induced degeneration and death . We found that P/M addition attenuated cell death evoked by beta-amyloid peptides (A beta(25-35) and A beta(1-40)) after 24 hr treatment and that this effect was blocked by alpha-ciano-3-hydroxycinnamate (CIN), suggesting that it requires mitochondrial pyruvate uptake . P/M supply to control and A beta-treated neuronal cultures increases cellular reducing power, as indicated by the ability to reduce the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) . The early increases in ROS levels, measured by dichlorofluorescein (DCF) fluorescence, and caspase-3 activity that follow exposure to A beta were notably reduced in the presence of P/M . These results place activation of caspase-3 most likely downstream of oxidative damage to the mitochondria and indicate that mitochondrial NAD(P) redox status plays a central role in the neuroprotective effect of pyruvate . Inhibition of respiratory chain complexes and mitochondrial uncoupling did not block the early increase in ROS levels, suggesting that A beta could initiate oxidative stress by activating a source of ROS that is not accesible to the antioxidant defenses fueled by mitochondrial substrates . Reprod Biomed Online, 2003 Jun, 6(4), 470 - 81 Towards a single embryo transfer; Gardner DK et al.; The delivery of a single, healthy child is the desired outcome of human assisted reproduction techniques . To attain this goal, there is an increasing movement toward single embryo transfer . The question is, therefore, at what stage to transfer the human embryo back to the uterus? Maximal implantation rates reported to date have come from the transfer of blastocysts (70% fetal heart rate) . In any given cycle of treatment the probability of conceiving a child will be further increased by the ability to cryopreserve those embryos not transferred . It is therefore proposed that the transfer of a single blastocyst is the best treatment for most patients, given the high implantation rates of fresh transfers, and that it is now possible to cryopreserve supernumerary blastocysts effectively . The next decision is how to culture the human embryo to the blastocyst stage . The use of sequential culture media, designed not only to allow for changes in nutrient requirements and metabolism as development proceeds, but also to minimize intracellular trauma, can facilitate the development of highly viable blastocysts . Sequential culture media have been evaluated against a single-step culture system . It has been shown that sequential media (G1/G2) produce more viable blastocysts than those embryos cultured in a single medium formulation (simplex optimized medium with elevated potassium and with amino acids, KSOM(AA)) throughout the preimplantation period . Furthermore, even if KSOM(AA) is used for embryo culture, it is essential that the medium be renewed after 48 h to alleviate the toxicity associated with ammonium build-up . Of great significance, embryos cultured in sequential media G1 and G2 have the same rate of development as embryos developed in vivo. Carbohydr Res, 2003 Jul 4, 338(14), 1517 - 21 Antitumor activities of heteropolysaccharides of Poria cocos mycelia from different strains and culture media; Jin Y et al.; Ten water-soluble heteropolysaccharide fractions were isolated from Poria cocos mycelia cultured from one wild and one cultivated strain in two identical culture media differing only in one component: either corn steep liquor or bran extract . The chemical compositions, including monosaccharide profile, protein content, and molecular mass M(w) of the mycelial polysaccharides were determined . Both the in vitro and in vivo antitumor activities of the heteropolysaccharides were evaluated and compared . The heteropolysaccharides from Poria cocos mycelia cultured with the wild strain in a medium containing corn steep liquor exhibited the highest antitumor activities against Sarcoma 180 in vivo. Carbohydr Res, 2003 Jul 4, 338(14), 1507 - 15 Effect of culture media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia; Jin Y et al.; Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively . Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II) . Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry . The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of alpha-D-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1-->3)-alpha-D-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1-->3)-beta-D-glucans . Interestingly, (1-->3) alpha- and (1-->3)-beta-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid . The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein . The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass . In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition. Cancer Sci, 2003 Mar, 94(3), 286 - 91 Enhanced sensitivity to cis-diamminedichloroplatinum(II) of a human carcinoma cell line with mutated p53 gene by cyclin-dependent kinase inhibitor p21(WAF1) expression; Satou M et al.; p53-mediated induction of p21(WAF1), a cyclin-dependent protein kinase inhibitor, is known to protect cancer cells from the cytotoxic effects of anti-cancer drugs or gamma-irradiation . Since the p53 gene is frequently inactivated in cancer cells, we examined whether p21(WAF1) expression may alter the sensitivity of cancer cells with mutated p53 gene to anti-cancer drugs . Cells of a colon cancer cell line DLD-1 were transfected with p21(WAF1) expression vector controlled by a tetracycline-repressable promoter and transfectants were cloned (Dp21-1) . p21(WAF1) expression induced by removal of tetracycline from culture media repressed cell proliferation and resulted in altered cell shape, suggesting induction of differentiation . Dp21-1 cells with p21(WAF1) expression were more sensitive to cis-diamminedichloroplatinum(II) (CDDP) (IC(50) value, 10 microM) than those without p21(WAF1) expression (IC(50), 22 microM) . Sensitivity to doxorubicin was not different between Dp21-1 cells with and without p21(WAF1) expression . DNA ladder formation was observed in Dp21-1 cells treated with CDDP, indicating that the enhanced sensitivity to CDDP involves apoptosis . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic protein revealed that subunit protein bands with M(r) 55 kDa and 44 kDa were markedly increased in cells with p21(WAF1) expression . By immunoblotting, these proteins were identified as c-Jun N-terminal kinase (JNK) 2 and p38 mitogen-activated protein kinase (MAPK) delta, respectively, both of which are believed to be involved in apoptosis induction by CDDP . These results suggest that p21(WAF1) may enhance the sensitivity of colon cancer cells with mutated p53 gene to CDDP, possibly through the JNK and p38 MAPK pathways. Cancer Sci, 2003 Mar, 94(3), 259 - 62 Glypican-3 is overexpressed in human hepatocellular carcinoma; Sung YK et al.; To identify candidate genes that could be used as diagnostic and therapeutic targets for hepatocellular carcinoma (HCC), we searched for the genes that are overexpressed in HCC by combining representational difference analysis and microarray . Genes such as glypican-3 (GPC3), insulin-like growth factor 2, long-chain fatty-acid-coenzyme A ligase 4, farnesyl diphosphate synthase were frequently identified in our screening . Northern blot analysis with these four genes confirmed their overexpression in HCC . Among them we found that GPC3 transcript is upregulated in six out of seven cases of HCC . Immunoblot and immunohistochemical staining using polyclonal anti-GPC3 antibodies further confirmed that GPC3 protein is indeed increased in HCC tumor samples . We also found that GPC3 is secreted into culture media from cell lines derived from HCC . We conclude that GPC3 is a good molecular marker for HCC. Exp Eye Res, 2003 Jul, 77(1), 85 - 92 Increased SPARC accumulation during corneal repair; Berryhill BL et al.; Keratocytes can become fibroblasts and myofibroblasts during corneal injury and wound healing . We used the in vitro bovine keratocyte repair model system, which involves culturing collagenase-isolated keratocytes in serum-free media and then adding serum or serum plus TGF-beta to the culture media to induce the fibroblast and myofibroblast phenotypes, respectively, to evaluate the synthesis of secreted products by the cells . Serum and serum plus TGF-beta rapidly induced the fibroblast morphology and alpha smooth muscle actin, a marker of myofibroblasts . Keratocytes cultured in serum and serum plus TGF-beta also increased the synthesis of several high molecular weight products (approximately 100kD and larger) and the accumulation of a 43kD protein shown to be osteonectin/SPARC by both sequencing tryptic peptides from the protein and by reaction with antisera to osteonectin/SPARC . Immunohistochemical staining of mouse corneas with antisera to SPARC seven days post-wounding also demonstrated an increased accumulation of SPARC in the regions undergoing repair . These results indicate SPARC accumulation is a marker for stromal repair. Zhonghua Yi Xue Za Zhi, 2003 Mar 10, 83(5), 412 - 6 {Mechanism of inhibition of tumor angiogenesis by Bletilla colloid: an experimental study}; Feng GS et al.; OBJECTIVE: To study the mechanism of inhibition of tumor angiogenesis by Bletilla colloid . METHODS: Human Hep-G2 hepatocellular carcinoma cells were cultured and treated with Bletilla colloid of different concentrations . Pure culture of Hep-G2 cells was used as control and pure culture medium without Hep-G2 cell was used as blank control . The concentration of vascular endothelial growth factor (VEGF) in the supernatant was detected by ELISA . The apoptosis and proliferation of the Hep-G2 cells were examined by flow cytometry . Cells of human endothelial cell line ECV-304 were cultured in the supernatant of culture media of Hep-G2 treated with Bletilla colloid of different concentrations . MTT method was used to observe the growth of the ECV-304 endothelial cells . Eighty rats were made animal model of transplanted Walker-256 hepatoma and then randomly divided into 4 groups of 20 rats 12 - 14 days after to undergo transarterial chemoembolization (TACE) treated with normal saline, 5-fluouracil (5-Fu), iodized poppy seed oil, and 5-Fu-Blettila microsphere respectively . Two weeks after, the rats were killed and the tumors were taken out . Immunohistological staining was conducted to detect the expression and localization of factor VIII, VEGF, and basic fibroblast growth factor (b-FGF) . The microvcascular density (MVD) was calculated by counting the factor VIII positive endothelial cells . RESULTS: There was no statistically significant difference in the apoptosis rate, proliferation rate and supernatant VEGF level between the control group and the Bletilla groups . The inhibition rates of ECV-304 endothelial cell growth were 57.6%, 66.7%, 86.4%, 87.5%, and 94.8% in the groups of Bletilla of the concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 micro g/ml respectively in a dose-dependent manner . The MVD of tumor was 59 +/- 34 per field in the 5-Fu-Blatilla group,significantly lower than those in the other 3 groups (F = 5.177, P = 0.003) . The expression of VEGF and the expression of b-FGF were not significantly different among the 4 TACE groups . CONCLUSION: Bletilla colloid inhibits angiogenesis after TACE, potentially, through inhibition of the binding of vascular endothelial growth factor to its receptor. J Vet Sci, 2003 Apr, 4(1), 73 - 8 Effects of protein source and energy substrates on the in vitro development of bovine embryos in a two-step culture system; Lim KT et al.; In this study, we examined the effects of a two-step culture system, which involves the use of different culture media for early cleavage and later stage embryos, on the in vitro development of bovine embryos . We also investigated the effect of glucose, phosphate and citrate on the in vitro early developmental period of bovine embryos in a two-step culture system . Moreover, the supplementation of different protein sources (BSA-V, BSA-FAF and FBS) during IVC did not affect the frequency of blastocyst development . Using two-step culture, embryos were cultured in protein-free media for an initial 5 days . This was then followed by the same culture media or an FBS supplemented media . The developmental rates of blastocysts in the FBS containing group were significantly higher than in the replaced with no serum containing group . Embryos cultured in mSOF supplemented with 1.5 mM glucose plus 1.2 mM phosphate were significantly inhibited . The inhibition of developmental competence by glucose plus phosphate was consistent with the existence of 0.5 mM sodium citrate . This study indicates that a two-step culture system, which applies different conditions for early cleavage embryos, i.e., serum-free media, vs . later stage embryos, with serum containing media, may be effective for in vitro production systems . In addition, the developmental competence of bovine embryos was depressed in the presence of glucose plus phosphate as compared to either alone or the absence of both . Therefore, the avoidance of this negative effect should allow more optimal conditions to be developed for in vitro production. Biochem Pharmacol, 2003 Jul 1, 66(1), 25 - 33 Involvement of nuclear transcription factor-kappa B in low-dose doxorubicin-induced drug resistance of cervical carcinoma cells; Yeh PY et al.; Administration of suboptimal doses of anticancer drugs not only fails to control tumor but often results in increased drug resistance of tumor cells . However, little is known about the effects of transient exposure to a minimally cytotoxic dose of chemotherapy on the development of drug resistance of the tumors . Previous studies have shown that upregulation of drug-exporter proteins (ATP-binding-cassette proteins) may be one of the key mechanisms involved in inducible drug resistance . In this study, we demonstrated that upregulation of NF-kappa B is another possible mechanism . SiHa cells were exposed to low-dose doxorubicin (100 nM; IC(30)) for 3 hr, and then were continuously cultured in drug-free culture media (designated as SiHa/DR cells) . SiHa/DR cells at up to 9 passages showed increased resistance to doxorubicin and cross-resistance to cisplatin . Results of quantitative real-time PCR and flow cytometry assay indicated that the increased drug-resistance in SiHa/DR cells was not due to upregulation of drug-exporter proteins or to the decrease of intracellular concentration of anticancer drugs . Both the basal and drug-induced NF-kappa B activity were shown to be increased in SiHa/DR cells by EMSA and NF-kappa B-driven luciferase reporter gene assay . Suppression of NF-kappa B activation by transfection of a dominant negative I kappa B alpha prevented the development of drug resistance, indicating that the upregulated NF-kappa B activity was positively correlated with the low-dose doxorubicin-induced drug resistance . These results suggest that even a transient exposure to a small dose of anticancer drugs may induce drug resistance in some cancer cells via upregulation of NF-kappa B activity. Clin Chem, 2003 Jul, 49(7), 1133 - 8 Disease-related metabolites in culture medium of fibroblasts from patients with D-2-hydroxyglutaric aciduria, L-2-hydroxyglutaric aciduria, and combined D/L-2-hydroxyglutaric aciduria; Struys EA et al.; BACKGROUND: D-2-Hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and the combined D/L-2-hydroxyglutaric aciduria (D/L-2-HGA) are poorly understood organic acidurias . To investigate the usefulness of cultured human skin fibroblasts for both diagnostic and research purposes, we measured disease-related metabolites in the cell culture medium . METHODS: We measured D-2-hydroxyglutarate (D-2-HG), L-2-hydroxyglutarate (L-2-HG), succinate, 2-ketoglutarate, and citrate in fibroblast cell medium by stable-isotope-dilution gas chromatography-mass spectrometry and glutamine, glutamic acid, and lysine with an amino acid analyzer . We used six cell lines from patients with D-2-HGA, two from patients with L-2-HGA, three from patients with D/L-2-HGA, and seven control cell lines . Culture medium was analyzed after a 96-h incubation period . RESULTS: Culture media from cell lines from D-2-HGA patients contained D-2-HG at concentrations 5- to 30-fold higher than media from controls, whereas the concentration of L-2-HG in media was not increased . Media from L-2-HGA cell lines showed a fivefold increase in L-2-HG compared with controls . Media containing fibroblasts from D/L-2-HGA patients contained moderately increased amounts of both D-2-HG and L-2-HG . For all cell lines, succinate concentrations in the blank medium were higher than after 96 h of incubation with the exception of two of three D/L-2-HGA cell lines . Media of D-2-HGA cell lines had 2-ketoglutarate concentrations that were 40% of that for controls . Glutamic acid concentrations in media of these cell lines were 60% lower than in controls . CONCLUSIONS: Cell culture media from fibroblasts from patients with D-2-HGA, L-2-HGA, or D/L-2-HGA contain increased amounts the corresponding 2-HGs, demonstrating the suitability of fibroblasts for both diagnosis of and research concerning 2-HGAs. J Biol Chem, 2003 Aug 29, 278(35), 32978 - 86 Epub 2003 Jun 18. Purification and characterization of recombinant, human acid ceramidase . Catalytic reactions and interactions with acid sphingomyelinase; He X et al.; Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA . The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity . The purified protein contained the same 13-(alpha) and 40 (beta)-kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using 14C- and BODIPY-labeled C12-ceramide as substrates . Deglycosylation studies showed that the recombinant enzyme contained mostly "high mannose" type oligosaccharides and that two distinct beta-subunits were present . Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the approximately 2-4-kDa molecular mass difference was likely due to C-terminal processing . The purified enzyme also catalyzed ceramide synthesis in vitro using 14C-labeled C12 fatty acid and sphingosine as substrates . Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be co-precipitated with acid ceramidase using anti-ceramidase antibodies . Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells . RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene . Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase . These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase. Kisaengchunghak Chapchi, 1988 Jun, 26(2), 95 - 106 {A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A . royreba}; Kim YK et al.; Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp . and Naegleria sp . has also been reported since the latter half of 1970's . However, no information on hydrolase activities in Acanthamoeba sp . is available . Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A . royreba . Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice . Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively . The results are summarized as follows: The mice infected with A . culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days . Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A . royreba did not . Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta-N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A . royreba . A . culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A . royreba did not show any specific cytotoxicity . About 80% of the target cells mixed with A . culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation . Hydrolase activities in A . culbertsoni cultured with the target cell line were assayed according to the culture time . The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates . Neutral proteinase activity in A . culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time . Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A . culbertsoni and non-pathogenic A . royreba, and that some hydrolase activities were detected remarkably higher in A . culbertsoni which revealed strong cytotoxicity to the target CHO cell line. Rheumatology (Oxford), 2003 Nov, 42(11), 1365 - 71 Epub 2003 Jun 16. Inhibitory effects of an anti-rheumatic agent T-614 on immunoglobulin production by cultured B cells and rheumatoid synovial tissues engrafted into SCID mice; Tanaka K et al.; OBJECTIVE: To clarify the pharmacological action of an anti-rheumatic agent T-614, we investigated its effects on immunoglobulin (Ig) production by cultured B cells and Ig secretion from synovial tissues of patients with rheumatoid arthritis (RA) using SCID mice engrafted with human RA tissue (SCID-HuRAg) . METHODS: Murine B cells were prepared from mouse spleen by a T-cell depletion method . The cells were cultured with lipopolysaccharide (LPS) and/or interleukin 4 (IL-4) in the absence or presence of T-614 . Human B cells were isolated from peripheral blood of healthy donors and the Ig production was induced by co-culture with autologous T cells and anti-CD3 antibody . SCID-HuRAg was prepared according to our previous method . T-614 was orally administered to the mice once daily for 4 weeks starting on the fourth week after the implantation . Then, peripheral blood was obtained and the implanted tissues were removed . Igs in the culture media or the sera were determined by enzyme-linked immunosorbent assay (ELISA) . RESULTS: In murine B-cell cultures, T-614 significantly decreased not only the IgM production stimulated with LPS but IgG1 production induced by LPS and IL-4 . Regarding human B cells stimulated with T cells, it also inhibited IgM and IgG production . In SCID-HuRAg mice, high concentrations of polyclonal human IgG were detectable in the sera of all mice . A significant decrease in the IgG level was observed in the T-614-treated group compared with the control group . CONCLUSIONS: We showed that T-614 inhibited Ig production by the cultured B cells and also decreased the high level of human IgG observed in SCID-HuRAg mice . These results may support its effect on plasma Ig in RA patients and provide insights into the mechanisms of its anti-rheumatic effect. Endocrinology, 2003 Jul, 144(7), 2957 - 66 Gonadotropin-releasing hormone signaling pathways in an experimental ovarian tumor; Chamson-Reig A et al.; Previous results showed that GnRH signaling is altered in cells from rat luteinized ovarian tumors (tumor group) because it did not activate the phospholipase C pathway, in contrast to control ovarian cells from superovulated prepubertal rats (SPO) . In the present work, alternate GnRH-induced second messengers such as phospholipase A(2) and phospholipase D activation, cAMP production, ERK1/2 phosphorylation, and the presence of G proteins were evaluated to determine GnRH mechanism of action in tumor cells . G proteins examined were present in both cell types . Buserelin, a GnRH agonist, (1, 10, and 100 ng/ml) increased phosphatidylethanol in SPO, indicating phospholipase D activation . Only 100 ng/ml buserelin induced a significant response in the tumor group . Buserelin (100 ng/ml) increased (3)H-arachidonic acid in culture media in SPO, indicating phospholipase A(2) activation; no effect was observed in the tumor group . Buserelin (100 and 1000 ng/ml) induced pertussis toxin-insensitive cAMP increases in both cell types, with similar potencies . In the tumor group, buserelin (100 ng/ml) inhibited human chorionic gonadotropin-induced cAMP and progesterone; this effect was protein kinase C (PKC) dependent (inhibited by GF109203X, a PKC inhibitor) . Buserelin (100 and 1000 ng/ml) induced ERK1/2 phosphorylation in both cell kinds . Buserelin-induced ERK1/2 activation was G(i/0) independent and PKC dependent . Only in the tumor group, buserelin-induced ERK1/2 activation was cAMP dependent (abolished by SQ 22536, the adenylyl cyclase inhibitor) . Furthermore, dibutyryl cAMP-induced ERK1/2 activation in the tumor group was PKC dependent (inhibited by GF109203X) . In conclusion, activation of phospholipases in tumor cells does not seem to mediate GnRH effects . GnRH signaling seems to involve adenylyl cyclase activation, PKC stimulation, and ERK1/2 phosphorylation. Di Yi Jun Yi Da Xue Xue Bao, 2003 Jun, 23(6), 608 - 10 {Effect of triglycerides on rat islet cell function in vitro}; Zhu ZY et al.; OBJECTIVE: To study the effect of triglycerides (TGs) on the function of in vitro cultured rat islet cells . METHODS: SD rat islet cells were isolated for monolayer cell culture and treated with TGs at different concentrations (0, 0.5, 2.5, 5.0, 10.0 mmol/L) for 72 h . Insulin level in the cell culture media was measured after glucose loading test, and the deposition of fat droplets in the islet cells observed by oil red O-staining . RESULTS: No obvious effect was observed after a 72-h incubation of the islet cells with TGs at low glucose level (2.8 mmol/L), while with the stimulation of high-level glucose (27.8 mmol/L), the insulin secretion was significantly inhibited by TGs of higher concentrations (5.0, 10.0 mmol/L) . The deposition of fat droplets was found in the islet cells after incubation with TGs, with TGs of higher concentrations . CONCLUSION: TGs induces deposition of fat droplets in islet cells in vitro, and may inhibit the insulin-secreting function of the cells. Immunology, 2003 Jul, 109(3), 398 - 406 Oestradiol regulation of antigen presentation by uterine stromal cells: role of transforming growth factor-beta production by epithelial cells in mediating antigen-presenting cell function; Wira CR et al.; We have previously shown that oestradiol treatment of ovariectomized rats for 3 days inhibits antigen presentation by uterine stromal cells at a time when oestradiol increases the numbers of antigen-presenting cells (APC) in the uterine stroma . In the present study, we found that oestradiol treatment for 1 day is sufficient to inhibit antigen presentation by stromal cells . To define the mechanism(s) of this inhibition, we examined the effect of cytokines and found that exogenous transforming growth factor-beta (TGF-beta) inhibits antigen presentation when stromal cells from saline- but not oestradiol-treated animals are incubated with ovalbumin (OVA)-specific T cells and OVA . In contrast, antigen presentation by uterine epithelial cells was not affected by TGF-beta . In other studies, the acute inhibitory effect of oestradiol (1 day) on stromal antigen presentation is fully reversed when anti-TGF-beta antibody is added to the culture media . When given for 3 days, oestradiol inhibition of antigen presentation is partially reversed by anti-TGF-beta antibody at a time when antibodies to tumour necrosis factor-alpha and interleukin-10 have no effect . To determine whether uterine epithelial cells produce TGF-beta, epithelial cells were grown to confluence on transwell inserts . Our findings indicate that uterine epithelial cells produce biologically active TGF-beta which is preferentially released basolaterally in the direction of underlying stromal cells . When oestradiol is given to ovariectomized rats 1 day before sacrifice, TGF-beta production by epithelial cells increases within 24 hr in culture, relative to saline controls . Taken together, these results suggest that oestradiol inhibition of stromal cell antigen presentation is mediated through the stimulatory effect of oestradiol on TGF-beta production by epithelial cells. J Pharmacol Exp Ther, 2003 Sep, 306(3), 846 - 54 Epub 2003 Jun 12. Prevention of ethanol-induced liver injury in rats by an agonist of peroxisome proliferator-activated receptor-gamma, pioglitazone; Enomoto N et al.; Agonists of peroxisome proliferator-activated receptor (PPAR)-gamma have been shown to reduce tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance . On the other hand, sensitization of Kupffer cells to lipopolysaccharide (LPS) and their production of TNF-alpha are critical for progression of alcoholic liver injury . This study was intended to determine whether pioglitazone, a PPAR-gamma agonist, could prevent alcohol-induced liver injury . Rats were given ethanol (5 g/kg b.wt.) and pioglitazone (500 microg/kg) once every 24 h intragastrically . Ethanol for 8 weeks caused pronounced steatosis, necrosis, and inflammation in the liver . These pathological parameters were diminished greatly by pioglitazone . Kupffer cells were sensitized to LPS after ethanol for 4 weeks as evidenced by aggravation of liver pathology induced by LPS (5 mg/kg) and enhancement of LPS (100 ng/ml)-induced intracellular Ca2+ concentration elevation in Kupffer cells . The parameters were diminished by treatment with pioglitazone . LPS-induced TNF-alpha production by Kupffer cells from the 4-week ethanol group was 3 to 4 times higher than control . This increase was blunted by 70% with pioglitazone . Gut permeability was 10-fold higher in the 4-week ethanol group, and pioglitazone treatment did not change the value . Inclusion of TNF-alpha in culture media of Kupffer cells enhanced CD14 expression, LPS-induced intracellular Ca2+ concentration response, and production of TNF-alpha . These results indicate that pioglitazone prevents alcoholic liver injury through abrogation of Kupffer cell sensitization to LPS. Surg Endosc, 2003 Nov, 17(11), 1818 - 22 Epub 2003 Jun 17. CO2 promotes plasminogen activator inhibitor type 1 expression in human mesothelial cells; Bergstrom M et al.; BACKGROUND: Previous observations have indicated that CO2 insufflation increases peritoneal plasminogen activator inhibitor type 1 (PAI-1) expression . METHODS: Primarily cultured human peritoneal mesothelial cells were exposed to either flowing or pressurized CO2 for 90 min . Unexposed cultures served as controls . Samples of cell culture media were taken at 0, 5, and 24 h after exposure to measure media pH, PAI-1, and tissue-type plasminogen activator (t-PA) protein release . Simultaneous samples were taken to measure PAI-1 and t-PA mRNA expression . RESULTS: Mesothelial cells exposed to flowing CO2 released more PAI-1 than those exposed to pressurized CO2 ( p < 0.001) and controls ( p < 0.001) . Cells exposed to flowing CO2 had an increased PAI-1 mRNA expression at 5 h . CONCLUSIONS: CO2 increased mesothelial cell PAI-1 expression involving a transcriptional mechanism . These findings might provide a mechanism for adhesion formation and cancer progression following laparoscopic surgery. Clin Oral Investig, 2003 Dec, 7(4), 198 - 205 Epub 2003 Jun 07. Effects of a vegetable extract from Lupinus albus (LU105) on the production of matrix metalloproteinases (MMP1, MMP2, MMP9) and tissue inhibitor of metalloproteinases (TIMP1, TIMP2) by human gingival fibroblasts in culture; Gaultier F et al.; This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture . LU105 was extracted from seeds of L . albus and is freely soluble in water . Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48 h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected . Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B) . LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9 . Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue . TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts . Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva. Osteoarthritis Cartilage, 2003 Jun, 11(6), 424 - 32 Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes; Dodge GR et al.; OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation . In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan . In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes . DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA . Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM . Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity . Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA . Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans . RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS . These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS . MMP-3 levels decreased (18-65%) as determined by Western blots . Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity . GS caused a dose-dependent increase (25-140%) in cell-associated GAG content . Chondrocytes obtained from 40% of OA patients failed to respond to GS . CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage . These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA. Comp Biochem Physiol B Biochem Mol Biol, 2003 Jun, 135(2), 231 - 9 Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes; Kim BH et al.; In vitro synthesis of vitellogenin (VTG), a female-specific protein, after estradiol-17 beta (E(2)) treatment was compared among different culture conditions using the hepatocytes of tilapia, Oreochromis mossambicus . VTG was measured by enzyme-linked immunosorbent assay . Comparison of Leibovitz's L-15 medium (L-15), Williams' medium E (WE) and Medium 199 (M199), which have been used for hepatocyte cultures in certain teleost fishes, showed that monolayer formation of the hepatocytes on the plate in WE and M199 was faster than in L-15 at the beginning of the culture . Morphological differences in the hepatocytes among the culture media were not evident by 96 h after culture . VTG synthesis in L-15 after E(2) treatment was higher than in WE and M199 . A concentration of NaHCO(3) at 5 mM in L-15 resulted in faster monolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM . Primary culture of the tilapia hepatocytes at 28 degrees C showed higher synthesis of VTG than at 23 and 33 degrees C . These results suggest that nutritional requirements are vitally different among species, and there are optimal ranges in the pH and the temperature in cultured hepatocytes. Plant Cell Rep, 2003 Apr, 21(8), 733 - 8 Epub 2003 Mar 13. High frequency plant regeneration from immature embryos of an elite barley cultivar (Hordeum vulgare L . cv . Morex); Chang Y et al.; An efficient plant regeneration system was developed for Hordeum vulgare L . 'Morex' barley, an important United States malting cultivar . The protocol was based on a series of experiments involving the sizes of immature embryos and the culture media . We found that the embryo size is critical for the establishment of embryogenic callus . Smaller embryos (0.5-1.5 mm) showed a much higher ability to produce embryogenic callus capable of regenerating green plants with fewer albinos than did the larger embryos (1.6-3.0 mm) . Either 3 mg/l 2,4-dichlorophenoxyacetic acid or dicamba in modified Murashige and Skoog's (MS) medium was optimum for the induction of embryogenic callus . The embryogenic callus maintained high regeneration during six subcultures in the callus induction medium . Efficient shoot regeneration was obtained on modified MS medium containing 0.5-1.0 mg/l 6-benzylaminopurine (BA) . Regenerated shoots were rooted on half-strength MS medium containing 0.2 mg/l IBA . Plants were successfully transferred to soil and grown to maturity in the greenhouse . This efficient plant regeneration system provides a foundation for generating transgenic plants of this important barley cultivar. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2003 Jun, 95(6), 739 - 45 Effects of root-end filling materials on fibroblasts and macrophages in vitro; Haglund R et al.; OBJECTIVE: The aim of this study was to investigate the effects of 4 root-end filling materials (mineral trioxide aggregate {MTA}, intermediate restorative material {IRM}, amalgam, and Retroplast) on cell growth, cell morphology, and cytokine (interleukin {IL}1beta and IL-6) production in mouse fibroblasts and macrophages . STUDY DESIGN: Millipore culture plate inserts with freshly mixed or set root-end filling material were placed into 6-well cell culture plates with already attached mouse fibroblasts or macrophages . Cells cultured with only the Millipore culture plate inserts served as a control . After a 3-day incubation, cell morphology was examined, and the total cell number per well was counted and analyzed by using 1-way analysis of variance . For cytokine assay, mouse macrophages were incubated in 24-well flat-bottom plates with set root-end filling material disks in the bottom . Cells cultured without the material disks served as negative controls, and cells cultured with lipopolysaccharides served as positive controls . After 24-hour incubation, culture media were collected for cytokine assay by using enzyme-linked immunosorbent assay . RESULTS: All root-end filling materials inhibited the cell growth of mouse fibroblasts and macrophages . There was no growth in the originally seeded cells in the fresh IRM, the fresh Retroplast, and the set IRM group . There was no difference between MTA and amalgam for cell growth either in the fresh material groups or in the set material groups . The total cell number in the set Retroplast group was significantly less than that in the set MTA group . Morphologically, MTA was characterized by denatured medium proteins and dead cells adjacent to the material, which were observed only in the fresh MTA group . There was no detected cytokine production in any of the tested material groups . CONCLUSION: All root-end filling materials inhibited cell growth, and none induced IL-1beta and IL-6 production. Mol Reprod Dev, 2003 Jul, 65(3), 269 - 77 Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media; Devgan V et al.; To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene . When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media . Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001) . However, percentages of morulae at 72 hr were comparable in all treatments . Developed blastocysts were better in M16 than in CZB or CZBG media . Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos . Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts . Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression . Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer . Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos . Exp Clin Endocrinol Diabetes, 2003 May, 111(3), 146 - 53 Targeted destruction of normal and cancer cells through lutropin/choriogonadotropin receptors using Hecate-betaCG conjugate; Zaleska M et al.; A recent approach to cancer treatment is destruction of malignant and non-malignant tumors by hormonally targeted lytic peptides . The presence of lutropin/choriogonadotropin (LH/CG) receptors has been confirmed in several cancer cells (e.g . breast, ovarian, and prostate) . In a series of experiments conducted in vitro, we have used a conjugate of the 23-amino acid lytic peptide Hecate and a 15-amino acid segment of beta-chain of CG . To test the hypothesis that Hecate-betaCG selectively destroys porcine granulosa and luteal cells, and Leydig cancer cell line (BLT-1) possessing LH/CG receptors, the conjugate was added to culture media at different concentrations of 0.5 to 10 micro M . Spleen cells and late passage of granulosa cancer cell line (KK-1) not-possessing LH/CG receptors were used as controls . The toxicity of Hecate-betaCG conjugate was concentration-dependent in all cell types but different among various cells . The toxicity of the conjugate to treated cells was closely correlated with the number of LH/CG receptors per cell . At low concentration (1 micro M), Hecate-betaCG was more cytotoxic to cells bearing LH/CG receptors than to controls (p < 0.01) . In contrast to cells possessing LH/CG receptors, cancer cell line KK-1 and spleen cells were sensitive only at concentration of 5 micro M (p < 0.001) . We conclude that Hecate-betaCG selectively kills cells expressing LH/CG receptors; its toxicity is dependent on the number of binding sites for LH/CG. Clin Lab Sci, 2002 Summer, 15(3), 131 - 5 Mycology at a distance; Koneman E et al.; GermWare Mycology is an image-rich, CD-ROM-based instruction divided into tutorial and reference programs . The tutorial program, designed for new students, provides only for sequential progress through each of the subject modules, so that each page of information is seen . In contrast, the reference program allows the more experienced learner with random and direct access to each facet of information . The aspergilli, the agents of chromomycosis, the dermatophytes, the dimorphic fungi, the hyaline molds, the dematiaceous molds, the yeasts, and the zygomyces are divided into separate modules . The tutorial program also includes an opening 'isolation procedures' module, in which details of specimen collection, culture media, and microscopic techniques are presented . The random access program includes system maps separating out each of the fungal species, and flow diagrams allowing an algorithm approach to species identifications . A global map is also included through which each fungal species can be directly accessed by the simple click of the mouse . Random access to information on the ecology, clinical presentations, pathology and therapy of the various mycotic diseases is also a feature of the reference program . A series of self-assessment exercises is included at the end of each module, with immediate 'pop-up' feedback to both correct and incorrect answers . The entire program includes over 2500 screens and over 700 color images and diagrams . GermWare Mycology is available through the Colorado Association for Continuing Medical Laboratory Education (CACMLE), who also can provide continuing education credits for individuals who complete a separate examination . For more information contact CACMLE at (303) 321-1734 or info@cacmle.org. J Lipid Res, 2003 Aug, 44(8), 1559 - 65 Epub 2003 Jun 01. GLP-1 stimulates glucose-derived de novo fatty acid synthesis and chain elongation during cell differentiation and insulin release; Bulotta A et al.; Glucagon-like peptide-1 (GLP-1, 7-36) is capable of restoring normal glucose tolerance in aging, glucose-intolerant Wistar rats and is a potent causal factor in differentiation of human islet duodenal homeobox-1-expressing cells into insulin-releasing beta cells . Here we report stable isotope-based dynamic metabolic profiles of rat pancreatic epithelial (ARIP) and human ductal tumor (PANC-1) cells responding to 10 nM GLP-1 treatment in 48 h cultures . Macromolecule synthesis patterns and substrate flow measurements using gas chromatography/mass spectrometry (MS) and the stable {1,2-13C2}glucose isotope as the tracer showed that GLP-1 induced a significant 20% and 60% increase in de novo fatty acid palmitate synthesis in ARIP and PANC-1 cells, respectively, and it also induced a significant increase in palmitate chain elongation into stearate utilizing glucose as the primary substrate . Distribution of 13C in other metabolites indicated no changes in the rates of nucleic acid ribose synthesis, glutamate oxidation, or lactate production . Tandem high-performance liquid chromatography-ion trap MS analysis of the culture media demonstrated mass insulin secretion by GLP-1-treated tumor cells . Metabolic profile changes in response to GLP-1-induced cell differentiation include selective increases in de novo fatty acid synthesis from glucose and consequent chain elongation, allowing increased membrane formation and greater insulin availability and release. Biol Reprod, 2003 Oct, 69(4), 1109 - 17 Epub 2003 May 28. Ammonium induces aberrant blastocyst differentiation, metabolism, pH regulation, gene expression and subsequently alters fetal development in the mouse; Lane M et al.; The presence of ammonium in the culture medium has significant detrimental effects on the regulation of embryo physiology and genetics . Ammonium levels build up linearly over time in the culture medium when media containing amino acids are incubated at 37 degrees C . Ammonium in the culture media significantly reduces blastocyst cell number, decreases inner cell mass development, increases apoptosis, perturbs metabolism, impairs the ability of embryos to regulate intracellular pH, and alters the expression of the imprinted gene H19 . In contrast, the rate of blastocyst development and blastocyst morphology appear to be normal . The transfer of blastocysts exposed to ammonium results in a significant reduction in the ability to establish a pregnancy . Furthermore, of those embryos that manage to implant, fetal growth is significantly impaired . Embryos exposed to 300 microM ammonium are retarded by 1.5 days developmentally at Day 15 of pregnancy . It is therefore essential that culture conditions for mammalian embryos are designed to minimize the buildup of ammonium to prevent abnormalities in embryo physiology, genetic regulation, pregnancy, and fetal development. Br J Pharmacol, 2003 May, 139(2), 457 - 63 Serotonin upregulates the activity of phagocytosis through 5-HT1A receptors; Freire-Garabal M et al.; 1 In this study, we investigated whether serotonin could regulate the in vitro activity of phagocytosis through 5-hydroxytryptamine or serotonin (5-HT(1A)) receptors . 2 Mouse peritoneal macrophages were cultured with serotonin and the activity of phagocytosis was assessed by the uptake of zymosan and latex particles added to the culture media . Specific binding of {(3)H}8-OH-DPAT and immunohistochemistry using an affinity-purified anti-5-HT(1A)-receptor antibody were assayed in the macrophages . In addition, we took advantage of the availability of pharmacological inhibitors of nuclear factor-kappaB (NF-kappaB) to explore its role in the regulation of the 5-HT(1A) receptor . 3 Serotonin increased the in vitro activity of phagocytosis in a dose-dependent manner . The 5-HT(1A) receptor agonist (+/-)-8-hydroxy-2-(di-n-propyl-amino)-tetralin (R(+)-8-OH-DPAT) reproduced these effects . Serotonin- or R(+)-8-OH-DPAT-induced increases in phagocytosis were blocked by the 5-HT(1A) receptor antagonist WAY100635 and the NF-kappaB inhibitor pyrrolidinedithiocarbamate . Moreover, mouse peritoneal macrophages expressed specific binding sites for {(3)H}8-OH-DPAT when cultivated in the presence of zymosan or latex beads . Immunohistochemistry confirmed the expression of the 5-HT(1A) receptor protein in the macrophages . 4 These results show that serotonin can upregulate the activity of peritoneal macrophages through 5-HT(1A) receptors. J Neuropathol Exp Neurol, 2003 May, 62(5), 509 - 19 Differential lipid peroxidation, Mn superoxide, and bcl-2 expression contribute to the maturation-dependent vulnerability of oligodendrocytes to oxidative stress; Bernardo A et al.; To understand the basis of oligodendrocyte (OL) susceptibility to oxidative injury, purified rat OL cultures at different stages of maturation were exposed to nitric oxide (NO) donors with fast or slow kinetics of release and to tert-butyl-hydroperoxide, a membrane-permeant organic hydroperoxide . OL precursors (pre-OL) displayed the highest vulnerability to both oxygen or nitrogen reactive species, whereas mature OLs were uniquely vulnerable to long-lasting levels of NO . Cell death occurred by necrosis as well as apoptosis associated with increased caspase-3 activity and, only in the case of pre-OLs, with a decreased expression of the anti-apoptotic protein bcl-2 . Pre-OLs were also more susceptible than mature OLs to lipid peroxidation, as measured by F2-isoprostane content in culture media . Finally, pre-OLs, but not mature OLs, expressed high levels of the mitochondrial scavenging enzyme Mn superoxide dismutase, suggesting that pre-OLs may efficiently convert anion superoxide into hydrogen peroxide and, paradoxically, be more predisposed than mature OLs to a toxic imbalance between hydrogen peroxide production and detoxification processes . These data suggest that susceptibility to lipid peroxidation, expression of the scavenging enzyme Mn superoxide dismutase and of the anti-apoptotic protein bcl-2, may contribute to the maturation-dependent vulnerability of OLs to oxidant injury. Biochemistry, 2003 Jun 3, 42(21), 6608 - 19 Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells; Panasiewicz M et al.; To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, {(3)H} labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C(8), C(12), C(14), C(16), C(18), C(22), C(24), C(18:1), C(22:1), or C(24:1) fatty acids (FAs), or C20 sphingosine acetylated or acylated with C(8) or C(18) FA were prepared and added to culture media . GM1s uptake by HL-60 cells was affected by the structure of their ceramides . Resistance to removal with trypsin and the stoichiometry of {(125)I} cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides . The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion . The GM1s with sphingosine acylated with C(14), C(16), C(18) C(22), or C(24) FAs were similarly abundant in DRMs . GM1s acylated with C(18:1), C(22:1), or C(24:1) were less abundant than those acylated with saturated FA of the same length . GM1s acetylated or acylated with C(8) FA were detected in DRMs in the lowest proportion . Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GM1s acetylated or acylated with C(8) or unsaturated FAs but not of GM1 acylated with C(18), C(22), or C(24) FAs . After cross-linking with CT B subunit, all GM1s were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion . DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions . The GM1s with sphingosine acetylated or acylated with C(18) or C(18:1) FAs occurred in these fractions in different proportions. J Cell Physiol, 2003 Jul, 196(1), 180 - 9 Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity; Kanatani M et al.; Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated . The present study was performed to investigate these issues . Increase in extracellular Pi concentration ({Pi}(e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) {1,25(OH)(2)D(3)} or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells . Increase in {Pi}(e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells . Increase in {Pi}(e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA . Increase in {Pi}(e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts . Increase in {Pi}(e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF . These results indicate that increase in {Pi}(e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells . It is also indicated that increase in {Pi}(e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts . Apoptosis, 2003 Jun, 8(3), 263 - 8 A novel assay for discovery and characterization of pro-apoptotic drugs and for monitoring apoptosis in patient sera; Biven K et al.; We have developed an apoptosis assay based on measurement of a neoepitope of cytokeratin-18 (CK18-Asp396) exposed after caspase-cleavage and detected by the monoclonal antibody M30 . The total amount of caspase-cleaved CK18 which has accumulated in cells and tissue culture media during apoptosis is measured by ELISA . The sensitivity is sufficient for use in the 96-well format to allow high-through-put screening of drug libraries . We here describe strategies allowing classification of pro-apoptotic compounds according to their profiles of induction of apoptosis in the presence of pharmacological inhibitors . The time course of induction of CK18 cleavage can furthermore be used to distinguish structurally similar compounds . We propose that compounds that induce rapid CK18 cleavage have mechanisms of actions distinct from conventional genotoxic and microtubuli-targeting agents, and we present one example of an agent that induces almost immediate mitochondrial depolarization and cytochrome c release . Finally, CK18-Asp396 cleavage products are released from cells in tissue culture, and presumably from tumor cells in vivo . These products can be measured in sera from cancer patients . We present evidence suggesting that it will be possible to use the M30-ELISA assay for measuring chemotherapy-induced apoptosis in patient sera, opening possibilities for monitoring therapy. Biotechniques, 2003 May, 34(5), 1074 - 8, 1080 Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer; Yamada K et al.; Recombinant lentiviral vectors stably transduce both dividing and nondividing cells . Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation . However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency . Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates . We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity . Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles . We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells . We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus . We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells. Cell Tissue Res, 2003 Jun, 312(3), 353 - 9 Epub 2003 May 23. Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin; Mori S et al.; Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis . Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials . In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin . Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin . HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin . Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG . Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media . These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials. Arterioscler Thromb Vasc Biol, 2003 Jul 1, 23(7), 1218 - 23 Epub 2003 May 22. Angiotensin II inhibits endothelial cell motility through an AT1-dependent oxidant-sensitive decrement of nitric oxide availability; Desideri G et al.; OBJECTIVE: The migratory capability of vascular endothelial cells plays a pivotal role in the maintenance of vessel wall integrity and is stimulated by nitric oxide (NO) . Angiotensin II increases NAD(P)H oxidase activity in endothelial cells, thereby promoting reactive oxygen species (ROS) generation . Because ROS can both reduce NO synthase activity and increase NO breakdown, thus impairing NO availability in endothelial cells, we evaluated the effect of angiotensin II on human vascular endothelial cell (HUVEC) motility . METHODS AND RESULTS: Angiotensin II dose- and time-dependently reduced HUVEC migration . Besides inhibiting HUVEC motility, angiotensin II altered intracellular glutathione redox status . The generation of ROS by cultured HUVECs was significantly increased by angiotensin II . Furthermore, angiotensin II reduced NO metabolite concentrations in culture media . The angiotensin II type 1 receptor antagonist candesartan cilexetil attenuated the inhibitory action exerted by angiotensin II on HUVEC motility, reversed the angiotensin II-induced increase in intracellular oxidative stress, and restored NO availability . Similar effects were exerted by the flavonoid inhibitor diphenylene iodinium and the antioxidant agent N-acetyl-L-cysteine . CONCLUSIONS: All together, our data demonstrate that angiotensin II inhibits HUVEC motility by reducing NO availability . Such reduction is due to an angiotensin II type 1 receptor-dependent increment in intracellular ROS generation. J Basic Microbiol, 2003, 43(3), 210 - 7 Effect of carbon source on alkaline phosphatase production and excretion in Aspergillus caespitosus; Guimaraes LH et al.; The effect of several carbon sources on the production of alkaline phosphatase by the thermotolerant Aspergillus caespitosus was analysed . The fungus released high levels of alkaline phosphatases into the medium after being cultured for long periods with xylan or industrial residues such as wheat raw and sugar cane bagasse in the culture media . In contrast, the alkaline phosphatase activities were found only intracellulary when the fungus was cultured in glucose-supplemented media . The pH of the medium likely affects the process of enzyme secretion according to the carbon source used . Addition of xylan or industrial residues in the culture medium stimulated the secretion of phosphatases . In contrast, media supplemented with glucose or disaccharides promoted retention of these enzymes into the cells . The subcellular location activities of alkaline phosphatases were studied using histochemical and immunochemical methods and showed that alkaline phosphatases were present in the mycelial walls and septa. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1999 Dec, 13(4), 348 - 51 {Optimization of productivity of Epstein-Barr virus membrane antigen in baculovirus infected insect cells}; Lu J et al.; OBJECTIVE: To probe factors influencing the expression level of in insect cells . METHODS: Insect cells at different growth phases and in different culture media were infected with recombinant baculovirus carrying EBV-MA gene, where the membrane anchor sequence was deleted . The expression level of EBV-MA was detected by immunodots assay and scanner analysis . RESULTS: Cells at the early exponential phase yielded more expressed recombinant EBV-MA than those at the late exponential and stationary phases . Infected cells supplemented with fresh medium at various phases of growth resulted in improved productivity of EBV-MA . Further experiments revealed that replacing fresh medium prior to infection and supplementing a mixture of glucose and glutamine after infection, the productivity could be improved dramatically . Comparing the productivity of EBV-MA expressed in sf9 cells with that in sf21 cells, no obvious difference was found . CONCLUSION: Consumption of nutrients and accumulation of metabolites in cell culture media were the important factors influencing the productivity of EBV-MA in insect cells. Neurosci Lett, 2003 Jun 5, 343(2), 109 - 12 Melatonin enhances lymphocyte proliferation and decreases the release of pituitary pro-opiomelanocortin-derived peptides in surgically traumatized rats; Huang YS et al.; The present study was undertaken to evaluate the effects of melatonin (MT) on spleen lymphocyte proliferation and release of pituitary pro-opiomelanocortin (POMC)-derived peptides in surgically traumatized rats . MT prevented the depression of lymphocyte proliferation induced by trauma in vivo and in vitro, and prevented the decrease of beta-endorphin and adrenocorticotropin in the pituitary in vivo, but dose-dependently inhibited the release of POMC-derived peptides from the pituitary in vitro . The culture media of the pituitaries derived from the traumatized rats inhibited lymphocyte proliferation of normal rats . These results suggest that MT can improve the trauma-induced depression of lymphocyte proliferation and inhibit the release of POMC-derived peptides . The neuroimmunomodulatory role of MT and POMC-derived peptides deserves further study. Bone, 2003 May, 32(5), 502 - 12 Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2; Hirata K et al.; We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors . In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein . Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts . In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier {poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)} and skin fibroblasts (PGS/SF complex) . Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2 . Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks . To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects . Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair . In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation . In addition, transplantation of these complexes was insufficient to induce bone repair . Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2. Anim Reprod Sci, 2003 Sep 15, 78(1-2), 71 - 84 The regulation of steroidogenesis by opioid peptides in porcine theca cells; Kaminski T et al.; The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488) . Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells . The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH . All delta receptor agonists depressed basal progesterone (P4) output . However, the influence of these agents on LH-treated cells was negligible . Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release . The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory . Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells . All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells . Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output . Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids . Basal secretion of E2 was also suppressed by kappa agonists . This inhibitory effect was not observed when the cells were additionally treated with LH . In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis. Ai Zheng, 2003 May, 22(5), 504 - 7 {Effect of cell cycle on telomerase activity of hepatoma cells and its relationship with replication of hepatitis B virus}; Yan SN et al.; BACKGROUND & OBJECTIVE: Previous studies have shown that the expression of telomerase activity is closely correlated with the formation and development of tumor cells . Furthermore, the cell cycle is associated with hepatitis B virus (HBV) replication level and telomerase activity . For further study their relationship, this experiment was designed to investigate the effect of serum deprivation or all-trans-retinoic acid(RA)on cell cycle of human hepatoma cells transfected by HBV DNA (HepG2 cell line) and the associations of cell cycle with telomerase activity and HBV replication . METHODS: Human hepatoma HepG2 cells were respectively treated with serum deprivation or RA . Cell cycle was analyzed using flow cytometry . Telomerase activity was determined quantitatively by TRAP-PCR-ELISA . HBV-DNA in culture media was determined using quantitative PCR and semiquantitative dot blot hybridization assay . HBsAg and HBeAg in cell culture media were measured using quantitative ELISA . RESULTS: RA treatment or serum deprivation inhibited the proliferation of HepG2 cells and the cells were arrested at G(0)/G(1) phase . The percentages of G(0)/G(1) phase of RA group and serum deprivation were 68.3% and 65.2%, respectively, while that of control group was 43.1% (P< 0.01) . The levels of telomerase activity also significantly decreased . The absorbance values that represented the telomerase activity of RA group and