Culture medium

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 serum deprivation group were 0.32 and 0.41, respectively, while that of control group was 1.34(P< 0.01) . In addition,HBV replication of HepG2 cells remarkably increased, which was shown as high products of HBV-DNA, HBsAg and HBeAg in culture media of RA group and serum deprivation group . The contents of HBV DNAs were 4.4x10(6), 5.1x10(6), and 1.2x10(6) copies/ml in RA group, serum deprivation group, and control group, respectively(P< 0.01) . The values of P/N of HBsAg were 3.5, 3.7, and 1.3 in RA group, serum deprivation group, and control group, respectively (P< 0.01) . The values of P/N of HBeAg were 19.8, 22.5, and 13.4 in RA group, serum deprivation group, and control group, respectively (P< 0.01) . CONCLUSION: Telomerase expression was associated with cell cycle in HepG2 cells . Telomerase was mainly expressed in S phase of cell cycle . HBV replication was also closely correlated with cell cycle, which increased in quiescent hepatocytes and decreased in proliferating hepatocytes.

Reprod Domest Anim, 2003 Jun, 38(3), 204 - 8
Manipulated mouse embryos as bioassay system for water quality control; Elsheikh AS et al.; Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K+ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D-O) water and Milli-Q system (M-Q) water . The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt) . The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source . The water source has no influence (p > 0.05) on development in EDTA-supplemented protein-free culture media, whereas in EDTA- and protein-free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M-Q water . The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01) . It worth mentioning that the rates of hatched blastocysts in protein-free culture media were very low (0-7.5%) . Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity . M-Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations . We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.

Parasite Immunol, 2003 Jan, 25(1), 1 - 7
Human anisakiasis: Diversity in antibody response profiles to the changing antigens in larval excretions/secretions; Hwang YK et al.; Anisakiasis is an infectious parasitic disease contracted by eating third stage larvae of Anisakis simplex (L3) carried by marine fishes . Human anisakiasis was researched for specific IgG with L3 excretory secretory products (ESP) . L3ESP were prepared by daily harvesting of culture supernatant from day 2 to day 5, using two kinds culture media of RPMI-1640 and phosphate buffered saline (PBS) . When the sera from persons diagnosed with anisakiasis by means of endoscopy were analyzed using indirect ELISA and Western blot, the sera was classified into four groups depending on specific antigen recognition patterns . In addition, the presence of a new antigen for L3, located at less than 13 kDa (AgLT13) was demonstrated in L3ESP with a modified Western blot condition . The production of AgLT13 was mainly found in L3ESP harvested both on day 2 and day 3, and that in PBS was predominant over that in RPMI-1640 . Among those sera, the high reactive sera to L3ESP-day two prepared with phosphate buffer in indirect ELISA recognized AgLT13, and 33% of the low reactive sera did so . These results indicate that the binding pattern of human L3 specific antibody is diverse depending on L3ESP preparations and that AgLT13 demonstrated with a Western blot condition is a specific antigen for L3.

Theriogenology, 2003 Jul, 60(2), 225 - 38
Identification, characterization and localization of three proteins expressed by the porcine oviduct; Buhi WC et al.; At estrus, the oviduct undergoes endocrine-induced changes which provide an essential microenvironment for maturation of gametes, fertilization and embryonic development . Several oviduct expressed proteins which interact with gametes or embryos, including the oviduct-specific, estrogen-dependent glycoprotein (OGP), have been identified and characterized . The objective of the present study was to identify, characterize and localize other proteins expressed by the porcine oviduct during estrus that may function in an autocrine or paracrine manner to enhance fertilization and embryonic development . Oviducts were collected during the estrous cycle or early pregnancy, flushed and divided into functional segments, and portions of the infundibulum, ampulla and isthmus were fixed for immunocytochemical analysis or cultured . Culture media was semi-purified by heparin-agarose affinity chromatography, proteins were transferred to polyvinylidene fluoride (PVDF) membrane after two-dimensional (2D)-SDS-PAGE and three different proteins were identified, excised and subjected to N-terminal amino acid analysis . These proteins were identified as complement component C3b, the carboxy-terminal propeptide of alpha 1 (III) procollagen (PIIICP), and the heavy chain variable region of IgA . Electrophoresis and fluorography of media from Days 0 to 12 of early pregnancy or the estrous cycle revealed both spatial and temporal expression of C3b and IgA heavy chain but not PIIICP by the oviduct . Further, all three proteins were identified in oviduct fluid by electrophoresis, immunoblot or immunoprecipitation analysis . Complement component C3b and IgA heavy chain were immunolocalized in all three oviduct segments on all days; however, temporal and spatial differences were demonstrated . Staining was greater in the infundibulum and during estrus for all three identified proteins . In summary, three proteins expressed by the oviduct at estrus and during early pregnancy were identified; characterization and localization suggest they may play a critical role in protecting the luminal environment, participating in ECM remodeling and gamete interactions.

Pediatr Crit Care Med, 2003 Apr, 4(2), 233 - 8
Perfluorooctyl bromide (perflubron) attenuates oxidative injury to biological and nonbiological systems; Rotta AT et al.; OBJECTIVE: To examine whether perfluorooctyl bromide (perflubron) is capable of protecting biological and nonbiological systems against oxidative damage through a mechanism independent of its known anti-inflammatory property . DESIGN: A controlled, in vitro laboratory study . SETTING: Research laboratory of a health sciences university . SUBJECTS: Rat pulmonary artery endothelial cell cultures (biological system) and linoleic acid in sodium dodecyl sulfate micelles (nonbiological system) . INTERVENTIONS: Rat pulmonary artery endothelial cells labeled with dichlorofluorescein diacetate and incubated with perflubron or culture media (control) were exposed to H2O2 . H2O2-induced fluorescence of dichlorofluorescein diacetate was measured as an index of intracellular oxidative stress . In another experiment, linoleic acid in sodium dodecyl sulfate micelles was exposed to various concentrations of the azo initiator 2,2'-diazo-bis-(2-amidinopropane) dihydrochloride (2, 4, 20, and 50 mM) in the presence or absence of perflubron . Malondialdehyde measurements were obtained as a marker of oxidative damage to linoleic acid . MEASUREMENTS AND MAIN RESULTS: Cell monolayers incubated with perflubron exhibited 66.6% attenuation in intracellular fluorescence compared with controls (p < .05) . Linoleic acid in sodium dodecyl sulfate micelles incubated with perflubron and exposed to 2, 4, 20, or 50 mM of 2,2'-diazo-bis-(2-amidinopropane) dihydrochloride showed less evidence of lipid peroxidation as indicated by lower malondialdehyde measurements at 240 mins (10.6%, 16%, 41%, and 14.2%, respectively) compared with controls . CONCLUSIONS: Perflubron attenuates oxidative damage to both biological and nonbiological systems . This newly recognized property of perflubron is independent of its anti-inflammatory properties.

Biomed Environ Sci, 2003 Mar, 16(1), 83 - 9
Modulation of isoflavones on bone-nodule formation in rat calvaria osteoblasts in vitro; Chang H et al.; OBJECTIVE: To observe the effects of two main isoflavones, daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro . METHODS: Osteoblasts obtained from newborn Sprague-dawley rat calvaria were cultured for several generations . The second generation cells were cultured in Minimum Essential Medium supplemented with ascorbic acid and Na-beta-glycerophosphate for several days, in the presence of daidzein and genistein, with or without the estrogen receptor antagonist ICI 182780 . Number of nodules was counted at the end of the incubation period (day 20) by staining with Alizarin Red S calcium stain . The release of osteocalcin, as a marker of osteoblast activity, was also determined on day 7 and day 12 during the incubation period . RESULTS: Compared with the control, the numbers of nodules were both increased by incubation with daidzein and genistein . 17 beta-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation that daidzein and genistein . The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein, as well as 17 beta-estradiol on day 7 and day 12 (day 12 were higher) . The estrogen receptor antagonist ICI 182780 completely blocked the genistein- and 17 beta-estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts . However, the effects induced by daidzein could not be inhibited by ICI 182780 . CONCLUSION: These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts . The effects, like those induced by 17 beta-estradiol, are mediated by the estrogen receptor dependent pathway . Daidzein also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts, but it is not, at least not merely, mediated by the estrogen receptor dependent pathway.

Endocrinology, 2003 Jun, 144(6), 2623 - 33
Ghrelin inhibits the development of mouse preimplantation embryos in vitro; Kawamura K et al.; Although ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues . Here, we investigated the expression of ghrelin and its receptor (GHS-R) in mouse oocyte and preimplantation embryos, and we examined the role of ghrelin in the regulation of early embryo development . Both ghrelin and GHS-R mRNAs were detected in morula or more advanced embryo stages . As for the origin of ghrelin, both ghrelin mRNA and protein were identified in the uterine endometrium . The levels of ghrelin in uterine fluid as well as plasma were significantly increased in fasting mice compared with animals with free access to foods . Addition of ghrelin to culture media inhibited the development of two-cell embryos to the hatched blastocysts, and the inhibitory effects of ghrelin were abolished by an antagonist for the GHS-R . In addition, ghrelin significantly decreased the number of total cells, inner cell mass, and trophectoderm cells in blastocysts . These observations suggest that ghrelin could inhibit the development of preimplantation embryos during fasting . Thus, ghrelin may act as a peripheral factor to avoid the excess metabolic demands imposed by pregnancy during malnutritional states.

J Biol Chem, 2003 Jul 18, 278(29), 26793 - 802 Epub 2003 May 12.
Transforming growth factor-beta1 produced by ovarian cancer cell line HRA stimulates attachment and invasion through an up-regulation of plasminogen activator inhibitor type-1 in human peritoneal mesothelial cells; Hirashima Y et al.; The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) and its specific inhibitor (plasminogen activator inhibitor type-1 (PAI-1)) could be involved in the pathogenesis of peritoneal dissemination . We showed previously that expression of uPA and PAI-1 in the human ovarian cancer cell line HRA can be down-regulated by exogenous bikunin (bik), a Kunitz-type protease inhibitor, via suppression of transforming growth factor-beta1 (TGF-beta1) up-regulation and that overexpression of the bik gene can specifically suppress the in vivo growth and peritoneal dissemination of HRA cells in an animal model . We hypothesize that the plasminogen activator system in mesothelial cells can be modulated by HRA cells . To test this hypothesis, we used complementary techniques in mesothelial cells to determine whether uPA and PAI-1 expression are altered by exposure to culture media conditioned by HRA cells . Here we show the following: 1) that expression of PAI-1, but not uPA, was markedly induced by culture media conditioned by wild-type HRA cells but not by bik transfected clones; 2) that by antibody neutralization the effect appeared to be mediated by HRA cell-derived TGF-beta1; 3) that exogenous TGF-beta1 specifically enhanced PAI-1 up-regulation at the mRNA and protein level in mesothelial cells in a time- and concentration-dependent manner, mainly through MAPK-dependent activation mechanism; and 4) that mesothelial cell-derived PAI-1 may promote tumor invasion possibly by enhancing cell-cell interaction . This represents a novel pathway by which tumor cells can regulate the plasminogen activator system-dependent cellular responses in mesothelial cells that may contribute to formation of peritoneal dissemination of ovarian cancer.

J Biol Chem, 2003 Jul 18, 278(29), 26788 - 92 Epub 2003 May 09.
Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate; Shiba T et al.; The proliferation of normal human fibroblast cells was enhanced by the addition of inorganic polyphosphate (poly(P)) into culture media . The mitogenic activities of acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) were also enhanced by poly(P) . A physical interaction between poly(P) and FGF-2 was observed, and FGF-2 was both physically and functionally stabilized by poly(P) . Furthermore, poly(P) facilitated the FGF-2 binding to its cell surface receptors . Because poly(P) is widely distributed in mammalian tissues, it may be a spontaneous modulator of FGFs.

Cell Tissue Res, 2003 Jun, 312(3), 313 - 8 Epub 2003 May 09.
Phenotype-dependent synthesis of transferrin receptor in rat alveolar epithelial cell monolayers; Widera A et al.; The iron carrier protein transferrin plays a prominent antioxidant and anti-bacterial role in the lower respiratory tract and is present at elevated concentrations in lung epithelial lining fluid relative to plasma . The level of transferrin receptor synthesis in primary cultures of rat alveolar epithelial cells (AECs) was investigated . Transferrin receptor was found to be synthesized early in AEC cultures with the alveolar type II cell-like phenotype . Cell-surface receptor localization was attenuated upon apparent transdifferentiation to the alveolar type I cell-like phenotype later in culture . Binding of (125)I-labeled transferrin to the receptor indicated that surface and total cellular transferrin receptor levels were decreased in the type I-like cells . Inclusion of keratinocyte growth factor (KGF) in culture media (10 ng/ml) resulted in retention of transferrin receptor localized to the basolateral surface . Transferrin-receptor-specific internalization of (59)Fe-transferrin was also limited to the basolateral surface of KGF-treated monolayers . These data suggest that alveolar type II (but not type I) cells express functional transferrin receptor in adult rat alveolar epithelium.

Int J Oncol, 2003 Jun, 22(6), 1241 - 5
Cell to cell contact required for bystander effect of the TNF-related apoptosis-inducing ligand (TRAIL) gene; Huang X et al.; We have previously reported that direct transfer of the TNF-related apoptosis-inducing ligand (TRAIL) gene resulted in an apoptotic bystander effect, and that this bystander effect was not transferable with cell culture media . To further characterize its mechanism we tested the bystander effect of TRAIL in the human ovarian cancer cell line DOV13, human lung cancer cell line A549, human hepatoma cell line Hepa G2, human breast cancer cell line MDA-MB231 and human colon cancer cell lines Lovo and DLD1 . The bystander target cells were transduced with an adenovector expressing the lacZ gene (Ad/CMV-LacZ), while the effector cells were transduced with an adenovector expressing the green fluorescent protein (GFP)/TRAIL fusion gene . Effector and target cells were then cocultured in the same well with or without effector and target cell contact . In all the cell lines tested, target cells were killed if effector and target cell contact was permitted . However, no bystander effect occurred if effector and target cell contact was prevented . Furthermore, the bystander effect and apoptosis induction of TRAIL was dramatically reduced if cells were seeded at a very low density . Moreover, in all the cell lines tested, no detectable soluble TRAIL was found in media from the TRAIL-expressing cell cultures . Together, our results demonstrated that release of soluble TRAIL from transfer of the wild-type TRAIL gene is minimal, and that the bystander effect of the TRAIL gene is mainly mediated by membrane-bound TRAIL on the surface of transduced cells.

Am J Physiol Heart Circ Physiol, 2003 Sep, 285(3), H1132 - 9 Epub 2003 May 08.
Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo; Katwa LC; Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI) . Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express alpha-smooth muscle actin . It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors . To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue . In cultured cells, we found: 1) expression of mRNA for ET precursor gene (ppET1), ECE-1, and ETA and ETB receptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETA and ETB receptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETB receptor blocker) . These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1 . Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.

Chemosphere, 2003 Jul, 52(2), 471 - 5
Do vesicle cells of the red alga Asparagopsis (Falkenbergia stage) play a role in bromocarbon production?
Marshall RA, Hamilton JT, Dring MJ, Harper DB.
The Rhodophyceae (red algae) are an established source of volatile halocarbons in the marine environment . Some species in the Bonnemaisoniaceae have been reported to contain large amounts of halogens in structures referred to as vesicle cells, suggesting involvement of these specialised cells in the production of halocarbons . We have investigated the role of vesicle cells in the accumulation and metabolism of bromide in an isolate of the red macroalga Asparagopsis (Falkenbergia stage), a species known to release bromocarbons . Studies of laboratory-cultivated alga, using light microscopy, revealed a requirement of bromide for both the maintenance and formation of vesicle cells . Incubation of the alga in culture media with bromide concentrations below 64 mgl(-1) (the concentration of Br(-) in seawater) resulted in a decrease in the proportion of vesicle cells to pericentral cells . The abundance of vesicle cells was correlated with bromide concentration below this level . Induction of vesicle cell formation in cultures of Falkenbergia occurred at concentrations as low as 8 mgl(-1), with the abundance of vesicle cells increasing with bromide concentration up to around 100 mgl(-1) . Further studies revealed a positive correlation between the abundance of vesicle cells and dibromomethane and bromoform production . Interestingly, however, whilst dibromomethane production was stimulated by the presence of bromide in the culture media, bromoform release remained unaffected suggesting that the two compounds are formed by different mechanisms.

Brain Res Brain Res Protoc, 2003 May, 11(2), 119 - 22
A rapid assay for glial cell line-derived neurotrophic factor and neurturin based on transfection of cells with tyrosine hydroxylase promoter-luciferase construct; Tanaka M et al.; Glial cell line-derived neurotrophic factor (GDNF), a potent survival and trophic factor for various neuronal cells, has been measured by assaying its bioactivity based on neurite outgrowth or cell proliferation . We describe herein a sensitive and simple non-radioactive quantitative bioassay for GDNF family proteins based on their ability to induce tyrosine hydroxylase (TH) gene expression . Human neuroblastoma SK-N-MC cells were stably transfected with expression constructs of c-ret and with a luciferase reporter gene driven by 2 kb of the rat TH gene promoter region . In the presence of GDNF, luciferase activity increased with 20 h of incubation . A dose-dependent increase in luciferase activity was observed in the presence of GDNF between 1 and 300 ng/ml . This assay was also applicable for the quantification of the bioactivity of neurturin, another member of the GDNF family showing an even more sensitive profile of dose dependency than GDNF . Typical culture media were applicable in this assay . This method can be easily applied to numerous samples of conditioned medium in a short time.

Hua Xi Yi Ke Da Xue Xue Bao, 2001 Mar, 32(1), 52 - 4
{Effects of retinoic acid on secretion of apolipoproteins A I, A II, B100, C III and E by cultured HepG2 cells}; Liu H et al.; OBJECTIVE: This study inquired into the role of retinoic acid in lipoprotein metabolism . METHODS: We observed the effect of retinoic acid on the secretion of apolipoproteins A I, A II, C III, B100 and E by cultured HepG2 cells . The apolipoprotiens contents in culture media were measured by radioimmunodiffusion assay (RID) kits developed by our research laboratory . 20-fold lyophilizely condensed culture media were used for the assays . RESULTS: Retinoic acid increased the secretion of apoA I, B100, C III and A II, and it inhibited the secretion of apoE . The effect of retinoic acid was strengthened in a dose-dependent manner . When the concentration of retinoic acid in cultured media was 2 x 10(-4) mol/l, the secretion of apoA I, A II, B100 and C III increased by 14.3% (P < 0.01), 23.8% (P < 0.05), 16.1% (P < 0.01) and 47.6% (P < 0.01) respectively, and the secretion of apoE decreased by 37.2% (P < 0.01) . CONCLUSION: These findings indicate that retinoic acid does not have a general effect on apolipoprotein secretion in HepG2 cells.

Reprod Fertil Dev, 2003, 15(1-2), 19 - 25
Superoxide dismutase affects the viability of thawed European mouflon (Ovis g . musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection; Berlinguer F et al.; This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f . or intracytoplasmatic sperm injection (i.c.s.i.) . Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin . After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD . Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin . At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e . 1 = immotile to 10 = maximum motility, as observed at the moment of thawing) . For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes . For i.c.s.i., semen derived from the same culture systems as that for i.v.f . was used, and incubated for 1 h under standard conditions . The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture . The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture . Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium . Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i . system fertilization rates were significantly higher in the presence of SOD . The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

J Exp Clin Cancer Res, 2003 Mar, 22(1), 125 - 34
Correlation of gangliosides GM2 and GM3 with metastatic potential to lungs of mouse B16 melanoma; Saha S et al.; Ten B16 mouse melanoma cell lines with increasing metastatic potential to lungs (B16LuF1 to B16LuF10) were generated by in-vitro & in-vivo selection technique starting with B16F1 melanoma cell line . The number of metastatic tumor nodules in lungs rose with increasing metastatic potential . Tumor cell gangliosides of B16LuF1 to B16LuF10 cell lines, analysed and compared with TLC, showed eight major ganglioside bands . Band1 to band6 corresponded with standard gangliosides GT1b, GD1b, GD1a, GM1, GM2 and GM3 respectively . Band7 and Band8 could not be identified . The concentration of total as well as individual ganglioside bands of B16LuF1 to B16LuF10 cells appeared to rise with increasing metastatic potential . Gangliosides from the plasma of these cell lines (B16LuF1 to B16LuF10) maintained in-vivo in C57BL/6 mice on TLC analysis gave eight major ganglioside bands, similar to those of cells . Plasma gangliosides appeared to rise with increasing metastatic potential . However, it was interesting to see that only band5 and band6 gangliosides in plasma increased almost linearly with increasing metastatic potential . The remaining six ganglioside bands in the plasma did not show such correlation . Band5 and Band6 gangliosides corresponded with standard gangliosides GM2 and GM3 respectively . Gangliosides of the spent culture media, secreted by these cell lines in-vitro in tissue culture also gave eight major ganglioside bands, similar to that of cells . Spent culture media gangliosides appeared to increase with increasing metastatic potential . However, concentration of only band5 and band6 gangliosides of spent culture media increased almost linearly with increasing metastatic potential, thus further confirming the role of band5(GM2) and Band6(GM3) gangliosides in regulating metastatic potential of B16-melanoma cells to lung.

Gynecol Endocrinol, 2003 Feb, 17(1), 37 - 44
Biosynthesis of MUC1 mucin in human endometrial adenocarcinoma is modulated by estradiol and tamoxifen; Paszkiewicz-Gadek A et al.; Polymorphic epithelial mucin MUC1 is expressed by most epithelial cancers, although free natural MUC1 antibodies are present in the circulation of healthy subjects as well as in that of cancer patients . The role of MUC1 mucin molecules in cancer cells of endometrium is not precisely known . The results reported here demonstrate that MUC1 biosynthesis in human endometrial adenocarcinoma cells (Ishikawa line) is stimulated by estradiol hormone and inhibited by tamoxifen, which was measured by {(14)C}threonine or {(3)H}glucosamine incorporation into MUC1 protein . Tamoxifen applied in combination with estradiol also inhibited this process, but pre-incubation of cells with estradiol resulted in a decrease in the inhibitory effect of tamoxifen . Electroblotting and reactions with antibodies against MUC1 core protein epitopes confirmed the presence of MUC1 in cell lysates and culture media of Ishikawa cells . Reactions with lectins showed the presence of oligosaccharide structures demonstrating antigen-T activity and the presence of sialic acid residues . The results confirm that there is downregulation of MUC1 expression in cancer culture cells treated with selective estrogen receptor modulators, which may be essential for reducing the migration of cancer cells and the metastatic properties of tumor cells.

J Neuropathol Exp Neurol, 2003 Apr, 62(4), 351 - 62
Neuron-conditioned media differentially affect the survival of activated or unstimulated microglia: evidence for neuronal control on apoptotic elimination of activated microglia; Polazzi E et al.; It is presently unknown what types of neuronal signals maintain microglial cells resting in the normal brain or control their activation in neuropathology . Recent data suggest that microglia activation induces apoptosis and that healthy neurons are controllers of the activation state and immune functions of microglia . In the present study we have evaluated, on microglial cells in cultures, whether neurons are able to affect their survival in resting conditions or upon activation with the bacterial endotoxin, lipopolysaccharide (LPS) . We report that neuron-conditioned culture media induced apoptosis of LPS-stimulated, but not of unstimulated, microglia . This effect was, however, only present when conditioned media had been exposed to differentiated neurons and not to immature ones, and was absent when glutamate receptors had been pharmacologically blocked in neuronal cultures . The effect was also blocked by heat-inactivation of the conditioned media . Media conditioned with either differentiated or undifferentiated cerebellar granule neurons positively affected the survival of unstimulated microglial cells when the standard concentration of fetal bovine serum (10%) was included in the culture media . Our results highlight the ability of differentiated neurons to maintain a controlled inflammatory state through production of factor(s) favoring the apoptotic elimination of activated microglia . They also suggest that immature neurons may, on the contrary, favor the survival of microglia during development.

Mycopathologia, 2002, 156(1), 41 - 5
Measuring diversity of endophytic fungi in leaf fragments: does size matter?
Gamboa MA, Laureano S, Bayman P.
Endophytic fungi inhabit living plant tissues without causing disease symptoms . Although abundant, the extent of their contribution to fungal biodiversity remains unclear . Since endophytic fungi are poorly known, especially in the tropics, current estimates of fungal species are probably conservative . Here we tested strategies for sampling endophytic fungi in tropical plants . We compared the number of fungi isolated from 400 mm2 leaf pieces that were divided into increasingly small fragments . Leaf pieces were surface-sterilized, cut into fragments and plated on culture media . For a given area, cutting leaf pieces into smaller fragments significantly increased the number of fungal morphospecies recovered . There was a strong linear relationship between size of fragments and number of fungi isolated . By extrapolation, an estimated 16 +/- 3 fungi could be recovered from a 2 x 2 cm leaf piece, using infinitely small fragments . This represents a large part of the fungal diversity estimated to exist in leaf endophytes in a population . We conclude that reducing the size and increasing the number of leaf fragments will increase the number of fungal species isolated . This strategy will help to estimate real values of endophytic fungal diversity.

Invest Ophthalmol Vis Sci, 2003 May, 44(5), 1859 - 65
Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells; McDermott AM et al.; PURPOSE: To investigate the expression of human beta-defensins (hBDs) by human corneal epithelium and determine the effects of proinflammatory cytokines on expression of human beta-defensin (hBD)-2 by human corneal epithelial cells (HCECs) in culture . METHODS: RNA was extracted from corneal epithelial cells scraped from cadaveric corneas and from cultured HCECs, and RT-PCR was performed to detect hBD-1, -2, and -3 mRNA . To study the effects of proinflammatory cytokines on expression of defensin, HCECs were cultured and then exposed to interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha for up to 36 hours, with a range of concentrations (0.01-100 ng/mL) . In some experiments, cells were pretreated with various cell signaling pathway inhibitors before the addition of IL-1beta . At the end of the incubations, the cells were harvested for RT-PCR and the culture media collected for the detection by immunoblot analysis of secreted defensin peptide . RESULTS: All epithelial tissue collected from cadaveric corneas expressed mRNA for hBD-1 . hBD-2 was detectable in two of eight donors corneas, whereas hBD-3 was detected in five . All primary cultures of HCECs expressed hBD-1 and -3 . A faint band for hBD-2 was detectable in three of eight cultures . Cultures of simian virus (SV)40-transformed HCECs always expressed hBD-1 and -3, but did not express hBD-2 under control conditions . IL-1beta and TNFalpha each stimulated the expression of hBD-2 in HCECs and were more effective in combination than alone . The effects of IL-1beta were concentration- (maximal at 10 ng/mL) and time-dependent (maximal at 12 hours and 24 hours for hBD-2 mRNA expression and protein secretion, respectively) . The upregulation of hBD-2 mRNA persisted for at least 24 hours after removal of IL-1beta . The NFkappaB inhibitors pyrrolidinedithiocarbamate (PDTC; 100 microM), caffeic acid phenethyl ester (CAPE; 90 microM), and MG-132 (25 microM), blocked IL-1beta-stimulated expression of hBD-2 . The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 microM) and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (25 microM) partially blocked (by 47% and 59%, respectively) the effect of IL-1beta . However, PD98059, an ERK inhibitor, had no effect . Genistein (50 microM) and dexamethasone (1 microM) also partially blocked (by 26% and 28%, respectively) the effect of IL-1beta . CONCLUSIONS: Human corneal epithelium expresses hBD-1 and -3 . hBD-2 is not typically present, but its expression can be stimulated by proinflammatory cytokines such as IL-1beta, acting through mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB pathways . Because IL-1 is known to be increased at the ocular surface after injury, the current observations provide a mechanism to explain the previous finding that hBD-2 is upregulated in regenerating corneal epithelium . Cytokine stimulation of hBD-2 expression most likely provides additional protection against infection and raises the possibility that this defensin in particular may be involved in the wound-healing response, per se.

Reproduction, 2003 May, 125(5), 657 - 65
Expression of lysyl oxidase and effect of copper chloride and ammonium tetrathiomolybdate on bovine ovarian follicle granulosa cells cultured in serum-free media; Kendall NR et al.; Subfertility that will respond to appropriate copper supplementation is a widespread problem in the UK dairy herd and, although characterized by reduced or absent oestrus and reduced conception rates, the exact cause remains unknown . The aim of this study was to investigate the expression of mRNA for the copper-dependent enzyme, lysyl oxidase, and the effect of copper and/or copper chelating thiomolybdates on FSH-induced differentiation of bovine granulosa cells cultured in serum-free media . Expression of lysyl oxidase mRNA was investigated using bovine specific primers and RT-PCR on cell lysates obtained from bovine granulosa cells cultured under optimum conditions for 0, 16, 24, 48, 96, 144 and 192 h . The effect of thiomolybdates and copper were investigated by supplementing optimized granulosa cell culture media with ammonium tetrathiomolybdate at 0, 0.1, 1, 10, 100 and 1000 micro g ml(-1), copper chloride at equimolar concentrations (0, 0.0516, 0.516, 5.16, 51.6, 516 micro g ml(-1)) or equimolar combinations of both media . Lysyl oxidase mRNA was expressed by the granulosa cells throughout the 192 h of culture . Thiomolybdate depressed oestradiol production in a dose-dependent manner at doses > 1 micro g ml(-1) and prevented the characteristic clumped appearance of granulosa cells in this serum-free system . Although the supplementation of copper alone had no effect at physiological doses, the use of the equimolar copper and thiomolybdate media ameliorated the effect of tetrathiomolybdates on both oestradiol production and cellular morphology . In conclusion, the results of the present study indicate that lysyl oxidase is expressed by granulosa cells, that thiomolybdates can prevent FSH-induced differentiation of bovine granulosa cells in vitro and that these effects can be reversed by copper supplementation . Overall, these data support the hypothesis that copper-responsive subfertility results from perturbation of the normal pattern of ovulatory follicle growth and development, an effect that may be mediated, at least in part, via lysyl oxidase activity.

Cell Tissue Res, 2003 Apr, 312(1), 31 - 40 Epub 2003 Mar 12.
Genetic and ultrastructural demonstration of strong reversibility in human mesenchymal stem cell; Tagami M et al.; We examined human bone marrow mesenchymal stem cells by applying real-time quantitative polymerase chain reaction (PCR) (RT-PCR) technology and electron-microscopic techniques . Our RT-PCR demonstrated that the values of peroxisome proliferation-activated receptor gamma2 (PPARgamma2) and lipoprotein lipase (LPL) mRNA dramatically increased according to adipogenic stimulation . The expressions of both PPARgamma2 and LPL mRNA were significantly reduced ( P<0.01) and almost disappeared after stimulation had ceased . The expressions of both genes, however, increased again by stimulation even though the cells were in a dedifferentiated state for a month . In the ultrastructural study, over 80% of the cells proceeded into morphologically well-developed adipocytes at the 12th day of induction/maintenance which were packed with lipid droplets and clusters . In the next process these lipid products were excreted from the cell bodies and the peripheral small parts containing numerous droplets were torn from the greater parts, which stuck tightly to each other and adhered to culture dishes . Adipocytes were not detected in the culture media during the final stage . The total cell number was equal to and over 90% of the cells dedifferentiated into fibroblast-like stem cells during the final maintenance period of 1 month . Furthermore the dedifferentiated cells quickly differentiated again into adipocytes by stimulation even if they were quiescent for 1 month . Thus we conclude that mesenchymal stem cells have strong reversibility from both the genetic and morphological points of view.

J Periodontol, 2003 Mar, 74(3), 277 - 88
The effects of progesterone on matrix metalloproteinases in cultured human gingival fibroblasts; Lapp CA et al.; BACKGROUND: Although pregnancy gingivitis is widely believed to result from elevated hormone concentrations, the mechanism(s) involved in the etiology of this condition remain unknown . Paradoxically, despite the apparent inflammation for a prolonged period, pregnancy gingivitis rarely progresses to periodontitis and usually resolves postpartum . We used several methods to test in vitro the hypothesis that the elevated progesterone levels of pregnancy might inhibit the production of some of the matrix metalloproteinases (MMPs) that are responsible for periodontal destruction . METHODS: Cultured human gingival fibroblasts (GF) were tested in phenol red-free, serum-free medium with or without the progestogen, medroxyprogesterone acetate (MPA; 10(-6) M), using interleukin-1beta (IL-1beta) to initiate immune responses and MMP production . These MMP responses were examined by macroarrays, reverse transcription-polymerase chain reaction (RT-PCR), zymograms, and enzyme-linked immunosorbent assay (ELISA) . RESULTS: Array analysis showed that pretreatment of GF with MPA reduced mRNA induction for MMPs-1, -3, and -10 in response to 6 to 8 hours incubation with IL-1beta . RT-PCR confirmed, that after 24 hours with IL-1beta , GF pretreated with MPA had undetectable levels of mRNA for MMPs-1, -2, -3, -7, -10, and -13 . Zymograms of culture media from this 24-hour period showed reduction in several proteolytic activities . Examination of such 24-hour media using ELISA for MMP-3 and pro-MMP-13 confirmed that secretion of these enzymes was upregulated by IL-1beta and modulated downward by pretreatment with MPA . CONCLUSIONS: Production by GF of numerous MMPs in response to IL-1beta was significantly reduced by progesterone . This steroidal modulation of proteolytic enzymes could help to explain why pregnancy gingivitis typically is not characterized by progression to periodontitis.

Reprod Biomed Online, 2002 May-Jun, 4(3), 294 - 302
Recent advances in the production of viable human embryos in vitro; Pool TB; The introduction of stress to embryonic blastomeres through inappropriate culture conditions results ultimately in the loss of viability . Retention of normal metabolic function in human preimplantation embryos, as well as those of other mammalian species, has been improved by the use of stage-appropriate culture media wherein energy substrates and amino acids are provided in a temporally evolving sequence . While the time dependence of nutrient exposure to embryos has received wide attention, spatial considerations in the embryonic microenvironment have received none . The manner in which media are presented to embryos, the rate at which media are changed, the rate at which cell products are removed and the macromolecular influences upon embryonic microenvironments have received far less attention in the experimental literature . Recent advances in micro-scale engineering allows for the rapid production of matrices containing culture channels slightly larger than the dimensions of preimplantation embryos . Microfluidic systems hold great promise for providing physical configurations yielding significantly reduced volumes but simultaneously providing control over the dynamics of media change and waste removal via fluid flow with time . Additionally, macromolecules may be presented from fixed sites in a minimum volume of solvent thus allowing us to test the importance of space and geometry in facilitating the physical and chemical effectiveness of the embryonic milieu.

Reprod Biomed Online, 2002 May-Jun, 4(3), 233 - 6
Recombinant human albumin as protein source in culture media used for IVF: a prospective randomized study; Bungum M et al.; Oocytes obtained from 85 women undergoing IVF/intracytoplasmic sperm injection (ICSI) treatment were randomly selected for culture in media containing either human serum albumin (HSA), (n = 42) or recombinant human albumin (rHA), (n = 43) . At the time of transfer, the two embryos with the overall best morphology were selected on day 2, 3 or 5 . Comparable rates of fertilization, cleavage, blastocyst formation and implantation were observed in both groups . The frequency of pregnancy and early pregnancy loss were also equal . It is concluded that culture media containing rHA seem to produce embryos of high quality comparable to media containing HSA . Therefore, rHA may replace HSA as a protein source in culture media for IVF and may reduce risks of prion contamination and transmission of plasma derived impurities.

In Vitro Cell Dev Biol Anim, 2002 Oct, 38(9), 523 - 8
Culture medium enhances semicarbazide-sensitive amine oxidase activity; Trent MB et al.; Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells . Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro . Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC) . In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO . In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media . Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement . Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages . Activity in rat plasma was reduced by approximately 25% in the presence of DMEM . The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement . This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.

Rev Saude Publica, 2003 Apr, 37(2), 242 - 6 Epub 2003 Apr 04.
{Isolation of potencially pathogenic free-living amoebas in hospital dust}; da Silva MA et al.; OBJECTIVE: To evaluate the occurrence of free-living amoebas of the genera Acanthamoeba and Naegleria is dust samples colleted in two hospitals . METHODS: One-hundred and thirty-two dust samples were collected in two hospitals in Brazil . Hospital collection sites were the following: intensive care unit, operation rooms, nursery, kitchen, emergency and infectious diseases isolation room . The isolation of the amoebas was performed in three culture media: non-nutrient agar inoculated with Escherichia coli, soy agar, and microculture in Giazzi-modified Pavlova's medium . The amoebas were identified according to morphological criteria . RESULTS: Amoebas of the genera Acanthamoeba and Naegleria were found in 45.5% of the samples, of which 41.6% were collected in the university hospital and 50% in the state hospital . Of all, 45.5% were positive for the genera Acanthamoeba and 3.8% for genera Naegleria . CONCLUSIONS: Potentially pathogenic free-living amoebas were seen in all sites of the two hospitals and Acanthamoeba was the most frequently isolated genera.

Ann Rheum Dis, 2003 May, 62(5), 440 - 3
Active leflunomide metabolite inhibits interleukin 1beta, tumour necrosis factor alpha, nitric oxide, and metalloproteinase-3 production in activated human synovial tissue cultures; Elkayam O et al.; BACKGROUND: Leflunomide is now an approved agent for the management of adult rheumatoid arthritis (RA) . Its active metabolite A771726 inhibits de novo pyrimidine biosynthesis . Although considered to be an immunosuppressive agent, its mechanism of action remains obscure . OBJECTIVES: Evaluation of the leflunomide active metabolite A771726 (LEF) effect on interleukin 1beta (IL1beta), tumour necrosis factor (TNFalpha), nitric oxide (NO), and stromelysin (metalloproteinase-3 (MMP-3)) production by activated human synovial tissue in culture . METHODS: Synovial tissue was obtained during surgery from patients undergoing total knee replacement owing to RA or osteoarthritis (OA), cut into small pieces, and cultured in Petri dishes with test materials as previously described . IL1beta, TNFalpha, NO, and MMP-3 were measured in the culture media after 48 hours incubation with different doses of LEF by methods previously described . RESULTS: LEF (0.3, 3, and 9 micro g/ml) inhibited IL1beta production in the presence of lipopolysaccharide (LPS; 3 micro g/ml) in a dose dependent manner (p<0.01) at LEF 0.3 micro g/ml . TNFalpha production in the presence of IL1beta (1 ng/ml) was also inhibited in a dose dependent manner (p<0.05 at LEF 0.3 micro g/ml) . NO and MMP-3 production in the presence of LPS (3 micro g/ml) was inhibited as well (p<0.01 at LEF 1 micro g/ml and at LEF 0.3 micro g/ml, respectively) . Synovial cell viability evaluated by the tetrazolium salt XTT was unaffected by the LEF concentration used . There was no qualitative difference in the response of OA and RA synovial tissue . CONCLUSION: Leflunomide may modulate the rheumatoid articular process by inhibition of local production of IL1beta, TNFalpha, NO, and MMP-3.

J Dermatol, 2003 Mar, 30(3), 181 - 8
Gamma interferon gene transfection efficiently inhibits proliferation of neurofibroma cell lines in vitro; Nakayama J et al.; Primarily isolated neurofibroma cell lines and a human dermal fibroblast cell line were transfected with human gamma interferon gene in vitro, and changes in the cell proliferation rates were investigated . The proliferation rates of both cell lines were remarkably suppressed after the gene transfection . In particular, the neurofibroma cell lines almost stopped proliferating five days after gene transfection . The tritium thymidine uptake of the fibroblast cell line was almost abolished three days after gene transfection . The culture media from both of the gene-transfected cell lines contained a measurable gamma interferon concentration as late as five days after transfection, this was detected by an enzyme-linked immunosorbent assay . These data suggest that gamma interferon gene therapy might be a possible treatment for intractable or inoperable neurofibromas in patients with von Recklinghausen's disease in the future.

Anticancer Res, 2003 Jan-Feb, 23(1A), 71 - 8
Activated eosinophils infiltrate MCF-7 breast multicellular tumor spheroids; Furbert-Harris PM et al.; BACKGROUND: Previous studies in our laboratory have shown that activated eosinophils and eosinophilic cell lines inhibit the in vitro growth of MCF-7 breast tumor cells . We have also shown that IL-4 and IL-5 partially inhibit MCF-7 growth in vitro . In this study MCF-7 multicellular tumor spheroids (MTS) were developed to study the effect of eosinophils and IL-4 on tumor growth . MATERIALS AND METHODS: Hypo- and hyperdense metrizamide density gradient fractions of eosinophils from peripheral blood of individuals with mild to moderate esoinophilia were co-cultured with 2-day-old MCF-7 MTS in medium containing bacto agar overlay at 37 degrees C . RESULTS: Light microscopic analyses revealed the attachment of eosinophil effector cells to the spheroid borders . Moreover, the culture media was greater than 90% devoid of effector cells . At six days post co-culture, very large spheroids were observed in both test and control dishes; however the necrotic cores in the co-cultures were more intense and larger than in the control . When MCF-7 tumor cells (1 x 10(6)) were pretreated with IL-4 at 0.5 ng/ml, there was a dramatic decrease in the number of spheroids formed . CONCLUSION: These data strongly indicate that cytokines like IL-4 and perhaps other eosinophil mediators are capable of killing and inhibiting tumor growth; they suggest that tumor infiltrating eosinophils can degranulate and release toxic inhibitory factors into the tumor milieu which destroy the surrounding tumor . These observations, along with the use of the eosinophil: MTS tumor model provide a unique model system for in depth studies of the role of eosinophils and the cytokines they produce in breast cancer and may offer potential therapeutic implications.

Connect Tissue Res, 2002, 43(4), 559 - 68
Treatment with insulin-like growth factor-1 increases chondrogenesis by periosteum in vitro; Mierisch CM et al.; The repair of defects in articular cartilage with hyaline tissue that is resilient to wear is a challenging problem . Fibrocartilaginous tissue forms in response to injury through the articular surface and degenerates under mechanical load . Because periosteum contains cells, which are capable of synthesizing cartilage matrix proteins, it has been used to repair defects in articular surfaces . Treatment of periosteal grafts with growth factors, particularly those that elicit chondrocyte gene expression, may improve tissue regeneration . Gene expression by periosteal explants in vitro was measured . Expression of type II collagen and aggrecan mRNA was increased in response to treatment with IGF-I . Furthermore, IGF-I treatment caused an increase in type II collagen and aggrecan mRNA that was time and concentration dependent . The effect of short and long-term (continuous) incubations was compared to determine if a pretreatment could be used to condition a graft for subsequent surgical use . Short-term incubation in vitro with IGF-I followed by incubation without IGF-I was nearly as effective at increasing expression of type II collagen and aggrecan mRNA as incubation for the same length of time with IGF-I present continuously in the culture media . Treatment with IGF-I also produced cell clustering and nodule formation which are indicative of chondrogenesis . These results suggest that pretreatment with IGF-I in vitro may enhance the effectiveness of a graft to produce hyaline cartilage in vivo . Whether the cellular and molecular changes we have observed can lead to the formation of tissue that withstands the mechanical forces exerted by weight bearing remains to be determined.

Nutrition, 2003 Apr, 19(4), 353 - 7
Effect of chromium on apolipoprotein A-I expression in HepG2 cells; Haas MJ et al.; OBJECTIVE: Chromium is a key micronutrient required for lipid and carbohydrate metabolism . Some but not all clinical trials have associated use of chromium supplements with improved insulin sensitivity and lipid profile including increased high-density lipoprotein cholesterol levels . METHODS: Because apolipoprotein A-I (apoA-I) is the principal protein of high-density lipoprotein, the molecular pathways underlying chromium-related changes in apoA-I expression were studied in a human hepatoma cell line (HepG2) transfected with full-length apoA-I promoter attached to the reporter chloramphenicol acetyl transferase gene . RESULTS: Exposure of these cells to different concentrations of chromium chloride (0, 0.5, 1.0, and 3.0 mM) resulted in a dose-dependent decrease in apoA-I promoter activity (chloramphenicol acetyl transferase activity expressed as a percentage of an internal control was 99.4 +/- 7.2% in control cells versus 87.6 +/- 5.0%, 73.4 +/- 2.3%, and 36.6 +/- 3.9%, respectively, P < 0.01) . Chromium chloride at 10 mM concentration was toxic and caused death in a large number of cells . Treating HepG2 cells with other minerals known to have insulin-sensitizing effects such as magnesium (1 mM), zinc (0.2 mM), and vanadyl sulfate (0.1 mM) significantly reduced apoA-I promoter activity in the presence and absence of 100 microU/mL of insulin . Northern blot analyses showed that the apoA-I mRNA content of cells treated with 0.2 mM of chromium chloride relative to G3PDH mRNA was not significantly increased compared with controls (0.652 +/- 0.122 versus 0.745 +/- 0.143, the ratio of apoA-I to glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA in control and chromium-treated cells, respectively) . Western blot analyses of proteins secreted in culture media indicated that neither chromium treatment of the HepG2 cells (858.0 +/- 151.4 arbitrary units) nor treatment with magnesium (1323.3 +/- 175.7) or vanadium (1102 +/- 78.7) significantly altered apoA-I concentrations compared with controls (1061.7 +/- 114.7) . However treatment of HepG2 cells with 0.2 mM of zinc significantly reduced apoA-I concentrations (291.0 +/- 29.2 versus 1061.7 +/- 114.7; P < 0.001) . CONCLUSIONS: Supraphysiologic concentrations of chromium and other minerals with known insulin-sensitizing activity may reduce apoA-I promoter activity in cultured cells . Whether similar changes may occur in vivo remains to be shown . However, these observations do not support the use of pharmacologic amounts of chromium supplementation to enhance the cardioprotective lipid profile.

Dev Biol, 2003 Apr 15, 256(2), 342 - 54
Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation; Barcroft LC et al.; Mammalian blastocyst formation is dependent on establishment of trophectoderm (TE) ion and fluid transport mechanisms . We have examined the expression and function of aquaporin (AQP) water channels during murine preimplantation development . AQP 3, 8, and 9 proteins demonstrated cell margin-associated staining starting at the 8-cell (AQP 9) or compacted morula (AQP 3 and 8) stages . In blastocysts, AQP 3 and 8 were detected in the basolateral membrane domains of the trophectoderm, while AQP3 was also observed in cell margins of all inner cell mass (ICM) cells . In contrast, AQP 9 was predominantly observed within the apical membrane domains of the TE . Murine blastocysts exposed to hyperosmotic culture media (1800 mOsm; 10% glycerol) demonstrated a rapid volume decrease followed by recovery to approximately 80% of initial volume over 5 min . Treatment of blastocysts with p-chloromercuriphenylsulfonic acid (pCMPS, > or =100 microM) for 5 min significantly impaired (P < 0.05) volume recovery, indicating the involvement of AQPs in fluid transport across the TE . Blastocysts exposure to an 1800-mOsm sucrose/KSOMaa solution did not demonstrate volume recovery as observed following treatment with glycerol containing medium, indicating glycerol permeability via AQPs 3 and 9 . These findings support the hypothesis that aquaporins mediate trans-trophectodermal water movements during cavitation .

Int J Surg Investig, 2000, 2(4), 253 - 7
Omega-6 fatty acids can inhibit Fas-mediated apoptosis in a human colorectal carcinoma cell line: a potential mechanism for escape from immune surveillance; Meterissian S et al.; The Fas/Fas ligand pathway may play an important role in the pathogenesis of colorectal carcinoma by allowing tumor cells to evade host immune defenses . Since dietary fats, in particular omega-6 fatty acids, facilitate tumor development their influence on the Fas/Fas ligand pathway needs to be elucidated . The purpose of this study was to determine the effect of membrane free fatty acid (FFA) alterations on Fas expression and sensitivity . Two human colorectal carcinoma cell lines (CX-1 and CCL-188) were grown in cell culture media supplemented with omega-3 (docosahexanoic acid) and omega-6 (linoleic acid) fatty acids . Membrane alterations were confirmed by gas chromatography (GC) . Cell surface Fas expression was determined with flow cytometry using an anti-Fas monoclonal antibody . Sensitivity to Fas mediated apoptosis was measured by now cytometric measurement of fragmented DNA stained with propidium iodide . Appropriate changes in the membrane FFA composition were found by GC . Cell surface Fas expression was unaffected in either cell line . Omega-3 fatty acids did not alter Fas sensitivity for either cell line compared to control (CX-1: 59.7%, +/- 5.4 vs 51.4% +/- 7.1 for control, CCL-188: 54.3% +/- 8.6 vs 51.2% +/- 4.8 for control) . Omega-6 fatty acids produced a significant decrease in Fas-mediated apoptosis (CX-1: 34.2% +/- 4.8 and CCL-188: 22% +/- 6.0, p < 0.05 vs control) . These data indicate that although membrane FFA alterations did not affect Fas expression, omega-6 fatty acids significantly decreased Fas-mediated apoptosis . This inhibitory effect may protect colorectal carcinoma cells from lymphocyte Fas-mediated cell death.

Biol Pharm Bull, 2003 Apr, 26(4), 438 - 47
Diesel exhaust particle-induced cell death of cultured normal human bronchial epithelial cells; Matsuo M et al.; We investigated the effect of diesel exhaust particles (DEPs) on normal human bronchial epithelial (NHBE) cells . Inclusion of DEPs in culture media was lethal to NHBE cells . NHBE cells are more susceptible to DEPs than other normal human lung cells, normal human pulmonary artery endothelial cells and normal human embryonic lung fibroblasts . DEP-induced cell death was mainly due to necrosis . Using the fluorescence probes diacetoxymethyl 6-carboxy-3',6'-diacetoxy-2',7'-dichloro-3',6'-dideoxydihydrofluorescinate and 4,5-diaminofluorescein diacetate, it was observed that hydrogen peroxide and nitrogen monoxide, respectively, were generated within DEP-exposed NHBE cells . DEP cytotoxicity increased or decreased with an increase or decrease in the cellular level of reduced glutathione (GSH) by treatment with L-buthionine-(R,S)-sulfoximine or ethyl reduced glutathionate, respectively . In addition, DEPs themselves decreased the cellular level of GSH in a dose-dependent manner . Upon exposure of NHBE cells to high concentrations of DEPs, their cellular GSH was depleted almost throughout . Further, the following agents decreased DEP cytotoxicity: 1) antioxidants 2,2,5,7,8-pentamethylchroman-6-ol, ebselen, and N,N'-bis(salicylidene)ethylenediaminomanganese(II) dihydrate (EUK-8); 2) iron ion-chelating agents disodium bathophenanthrolinedisulfonate and desferrioxamine mesylate; 3) nitrogen monoxide synthase inhibitors N(G)-nitro-L-arginine methyl ester hydrochloride and N(G)-methyl-L-arginine acetate salt; and 4) an endocytosis inhibitor quinacrine . On the basis of these observations, the mechanism of DEP cytotoxicity toward NHBE cells is discussed.

Glycobiology, 2003 Jul, 13(7), 539 - 48 Epub 2003 Apr 02.
N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system; Choi O et al.; N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined . The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter . This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection . N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS . Structures of 11 glycans (88.8% of total N-glycans) were elucidated . The glycan analysis revealed that the most abundant glycans were Man1-3(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%) . There was only approximately 6% of high-mannose type glycans identified . Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue . However alpha(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected . Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.

Life Sci, 2003 Apr 25, 72(23), 2581 - 90
Release of prostaglandin E-2 in bovine brain endothelial cells after exposure to three unique forms of the antifungal drug amphotericin-B: role of COX-2 in amphotericin-B induced fever; McGuire TR et al.; Common formulations of amphotericin-B include a deoxycholate colloidal suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (l-Amph) . The clinical incidence of infusion related fever is highest with d-Amph, intermediate with Ablc, and lowest with l-Amph . In the present study, we measured the activation of cyclooxygenase-2 (COX-2) and subsequent release of prostaglandin E-2 (PgE-2) from brain microvessel endothelium treated with these three formulations of amphotericin-B . Primary cultured bovine brain microvessel endothelial cells (BBMEC) were exposed to d-Amph, Ablc and l-Amph at concentrations that can be achieved in the plasma of patients receiving the drug . Media samples from the cells were collected and analyzed for PgE-2 . Release of PgE-2 from BBMEC monolayers treated with l-Amph was similar to cells receiving culture media alone . In contrast, Ablc and d-Amph caused significantly greater release of PgE-2 from BBMEC monolayers compared to controls receiving culture media alone . PgE-2 release after d-Amph treatment was similar in magnitude to that observed with bacterial lipopolysaccharide (LPS) . Western blot analysis indicated significant induction of COX-2 expression in BBMEC following LPS, Ablc or d-Amph treatment . Furthermore, PgE-2 release following exposure of BBMEC monolayers to either LPS or the various amphotericin-B formulations was reduced by the addition of the selective COX-2 inhibitor, NS-398 . These studies indicate that amphotericin-B induces COX-2 expression in brain microvessel endothelium resulting in release of fever producing PgE-2 . The magnitude of PgE-2 release from BBMEC following exposure to various amphotericin-B formulations mirrors the clinical observations regarding amphotericin-B induced fever and serves as initial support for the clinical use of COX-2 inhibitors to reduce amphotericin-B fever.

J Int Acad Periodontol, 2000 Jan, 2(1), 14 - 8
A comparison of the effects of two kinds of glass-ionomer cement on human gingival fibroblast attachment, proliferation and morphology in vitro; Yan F et al.; The aim of this study was to compare the response of cultured human gingival fibroblasts to two kinds of glass-ionomer cement in vitro with regards to understanding the biocompatibility of these materials and their suitability for use as subgingival restorative materials . The glass-ionomer cements tested were Ketac Fil, and GC Fuji II . These cements were mixed according to the manufacturers' directions and condensed into tissue culture wells or cavities prepared on the root surfaces of extracted teeth . Healthy root slices and untreated tissue culture wells were used as controls . Several features such as attachment, proliferation and morphology of cells were studied . The results indicated that cells could not attach to the glass-ionomer cements unless they had been washed with distilled water and tissue culture media . No significant difference in initial attachment (1.5h) and long term attachment (48h) between the groups tested was noted . No significant difference was observed between levels of proliferation for human gingival fibroblasts grown on Ketac Fil, Fuji II, root slices and culture wells . No significant alterations of the cytomorphology could be detected in glass-ionomer cement compared with root slices . These results indicate that provided the surface is adequately washed, Ketac Fil, and Fuji II may be acceptable subgingival root surface restorative materials.

Biol Trace Elem Res, 2002 Winter, 90(1-3), 117 - 42
Impact of boron deficiency on Xenopus laevis: a summary of biological effects and potential biochemical roles; Fort DJ et al.; The toxicity of boron has been understood for many years . However, limited data currently exist concerning the nutritional essentiality of B in chordates . Results from an ongoing research program evaluating the nutritional essentiality of B in the South African clawed frog, Xenopus laevis, found that X . laevis fed a low-B diet in a low-B culture media produced a substantially higher number of necrotic eggs and fertilized embryos than frogs fed a boron-sufficient diet . Markedly decreased embryo cell counts at mid-blastula transition and an increased frequency of abnormal gastrulation were also noted in embryos from adult frogs fed the B-deficient diet . By 96 h of development, none of the larvae collected from the B-deficient adults and maintained in low-boron culture media developed normally . Reproductive effects associated with B deficiency in female Xenopus included ovary atrophy, oocyte necrosis, and incomplete oocyte maturation . In males, a decrease in testis weight and sperm count was noted . These studies suggest that these adverse effects resulting from B deficiency could be found during gametogenesis, gamete maturation, embryonic development, and larval maturation . The studies also confirmed that B deficiency was capable of interrupting the X . laevis life cycle . Additional studies evaluating the role of B in the thyroid axis and the oocyte plasma membrane progesterone receptor provide the first line of direct evidence for a biochemical role of boron in X . laevis . Combined together, this research program provides firm evidence that B is nutritionally essential in X . laevis.

J Periodontol, 2003 Feb, 74(2), 196 - 201
Are cytokines linked to collagen breakdown during periodontal disease progression?
Ejeil AL, Gaultier F, Igondjo-Tchen S, Senni K, Pellat B, Godeau G, Gogly B.
BACKGROUND: Evidence of the role of cytokines produced by resident and inflammatory cells during inflammation is well established . The aim of this study was to quantify in healthy and diseased human gingiva the area fraction (AA%) occupied by collagen fibers and the amount of cytokines such as interleukin (IL)-1beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) to investigate a possible correlation between such cytokines, collagen degradation, and the gingival index . METHODS: Gingival tissue specimens from 6 healthy controls (group 1), 6 patients with mild gingival inflammation (group 2), 6 patients with moderate gingival inflammation (group 3), and 6 patients with severe gingival inflammation (group 4) were cultured for 72 hours, and the cytokines present in the culture media were quantified using an enzyme-linked immunosorbent assay (ELISA) . Paraffin gingival sections from the 24 subjects were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis . RESULTS: The present study revealed significant differences (P < 0.05) between means of AA% in group 1 (53%), group 2 (41%), group 3 (39.5%), and group 4 (35%) for collagen fibers . Compared to controls, there were significant increases of IL-1beta (groups 3 and 4), IL-6, and TNF-alpha (group 3); a significant decrease of IL-4 (groups 2, 3, and 4) and TGF-beta (groups-2 and, 3); and no change of EGF . The collagen AA% was significantly correlated with the amounts of IL-4 and TGF-beta, and significantly inversely correlated with the amounts of IL-1beta for all 3 inflamed groups and IL-6 and TNF-alpha for groups 2 and 3 . CONCLUSION: The present study showed that EGF was not changed in inflamed gingival tissue and that IL-1beta and IL-4 were particularly and intensively correlated with collagen loss . These 2 cytokines could be markers of clinical severity during active periodontitis.

J Periodontol, 2003 Feb, 74(2), 188 - 95
Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy and diseased human gingiva; Ejeil AL et al.; BACKGROUND: The purpose of this study was to quantify the amount of matrix metalloproteinases such as MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 and tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 expressed by human gingival explants in culture media and the area fraction (AA%) of gingival collagen fibers according to the degree of inflammation, to investigate a possible correlation between these enzymes and collagen loss . METHODS: Gingival tissue specimens from 6 healthy controls (group 1), 17 patients with mild gingival inflammation (group 2), 10 patients with moderate gingival inflammation (group 3), and 9 patients with severe gingival inflammation (group 4) were placed in organ culture for 3 days . The MMPs and TIMPs in the culture media were quantified using zymography, dot blotting, and Western blotting . Paraffin gingival sections were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis . RESULTS: The AA% occupied by collagen fibers significantly decreased from group 1 (53%) to group 4 (35%) . The decrease in collagen fibers was inversely correlated with the significant increase in MMP-1, MMP-9, and MMP-13 (dot blotting analysis), with the increase of the active form of MMP-2, and with the active form and proform of MMP-9 (zymography analysis) . CONCLUSION: The present study showed that metalloproteinases, particularly MMP-2, MMP-9, MMP-1, and MMP-13, are involved in the gingival extracellular matrix degradation during periodontitis.

J Biomater Sci Polym Ed, 2003, 14(2), 139 - 55
Enhanced production of carcinoembryonic antigen by CW-2 cells cultured on polymeric membranes immobilized with extracellular matrix proteins; Hara M et al.; Cell growth and the production of carcinoembryonic antigen (CEA) were investigated in human colorectal adenocarcinoma tumor (CW-2) cells cultured on extracellular matrix (ECM) protein membranes, heat-treated poly(vinyl alcohol-co-ethylamine) (PVA-EA) membranes, and PVA-EA membranes containing immobilized ECM proteins . The highest concentration of CEA was found in the cell culture media of CW-2 cells on collagen (COL)-immobilized PVA-EA membranes . This is explained by the flexible mobility of COL on the COL-immobilized PVA-EA membranes causing a specific cell response for the production of CEA . An inverse relationship was observed between either the cell density or the CEA concentration in the cell culture media and the amount of fibronectin (FN) adsorbed on the COL-immobilized membranes . The CEA concentration in the cell culture media was directly related to the cell density, which, in turn, is inversely related to the amount of FN secreted by CW-2 cells . These findings indicate that cells tend to attach to the surface by secreting ECM proteins such as FN when they are grown on substrates that provide weak cell attachment.

J Anim Sci, 2003 Mar, 81(3), 753 - 64
Selective protein loss in lactating sows is associated with reduced litter growth and ovarian function; Clowes EJ et al.; This study was designed to test the degree of protein loss that may be sustained by lactating sows before milk biosynthesis and ovarian function will be impaired . First-parity Camborough x Canabrid sows were allocated to receive isocaloric diets (61 +/- 2.0 MJ of ME/d) and one of three levels of protein intake in lactation: 1) 878 g of CP and 50 g of lysine/d (n = 8), 2) 647 g of CP and 35 g of lysine/d (n = 7), or 3) 491 g of CP and 24 g of lysine/d (n = 10) . Every 5 d during a 23-d lactation, sow live weight, backfat depth, and litter weight were recorded, and a preprandial blood sample was collected . Milk samples were collected on d 10 and 20 of lactation . Sows were slaughtered on the day of weaning, and liver and ovarian variables were measured . Lower dietary protein intakes elicited progressively larger live weight losses during lactation (-13, -17, and -28 +/- 2.3 kg; P < 0.001), but similar and minimal backfat losses (-1.3 +/- 0.29 mm) . Approximately 7, 9, and 16% of the calculated body protein mass at parturition was mobilized by d 23 . Lactation performance did not differ among treatments until d 20, at which time approximately 5, 6, and 12% of the calculated protein mass at parturition had been lost . The milk protein concentration on d 20 of lactation reflected the amount of body protein lost, and was lowest (P < 0.05) in sows that lost the most protein . After d 20, piglet growth rate decreased (P < 0.05) in a manner related to the amount of body protein lost . At weaning, ovarian function was suppressed in sows that had mobilized the most body protein; they had fewer medium-sized follicles (> 4 mm; P < 0.05), their follicles contained less (P < 0.01) follicular fluid, and had lower estradiol (P < 0.05) and IGF-I (P < 0.10) contents . Culture media containing 10% pooled follicular fluid (vol/vol) from high-protein-loss sows were less able to support nuclear and cytoplasmic maturation of oocytes in vitro, evidenced by more oocytes arrested at metaphase I (P < 0.05) and showing limited cumulus cell expansion (P < 0.06) . Plasma insulin and IGF-I concentrations did not seem to be related to the observed differences in animal performance . Our data suggest that no decline in lactational performance or ovarian function when a sow loses approximately 9 to 12% of its parturition protein mass . However, progressively larger decreases in animal performance are associated with a loss of larger amounts of body protein mass at parturition.

Mol Reprod Dev, 2003 May, 65(1), 51 - 6
TUNEL analyses of bovine blastocysts after culture with EGF and IGF-I; Sirisathien S et al.; Experiments were carried out to investigate the beneficial effects of IGF-I or EGF on bovine embryo development in chemically defined embryo culture media and resultant incidences of nuclear DNA fragmentation as an indication of embryo quality . Presumptive IVF zygotes were randomly cultured in either control (with no added growth factor) or treatment groups, i.e., with 50 ng/ml IGF-I (experiment 1) or 5 ng/ml EGF (experiment 2) . IGF-I supplemented to culture media significantly improved proportions of blastocysts from oocytes inseminated compared to untreated controls (38.0% vs . 28.5%) . Only embryos reaching the blastocyst stage on day 8 showed significant effects of IGF-I treatment by resulting in higher blastocyst cell numbers (162 vs . 141) and lower percentages of TUNEL positive nuclei (2.1% vs . 3.3%) when compared to controls . Blastocyst development from oocytes was also improved by EGF supplementation compared to untreated controls (38.5% vs . 30.7%) . Cell numbers of either day 7 or day 8 blastocysts were not affected by EGF treatment, nor were percentages of TUNEL positive nuclei when compared with controls . Similar proportions of parthenogenetically activated oocytes developed to blastocysts as for inseminated oocytes (28.8%) . Parthenogenetic blastocysts contained fewer cells (93) and an increased percentage of TUNEL positive nuclei (5.7%) than were found for IVF embryos .

Cell Death Differ, 2003 Jan, 10(1), 134 - 41
Neutralization of TRAIL death pathway protects human neuronal cell line from beta-amyloid toxicity; Cantarella G et al.; Here we report that a novel member of the TNF-alpha family, TNF-related apoptosis-inducing ligand (TRAIL), contributes substantially to amyloid-induced neurotoxicity in human SH-SY5Y neuronal cell line . Involvement of TRAIL in the amyloid-induced cell death is supported by cDNA array, Northern blot, and Western blot data, demonstrating increased TRAIL expression after treatment of the cells with a neurotoxic fragment of amyloid protein (betaAP) . TRAIL was also found to be released in the culture media after betaAP treatment with a time-course overlapping to contents of the intracellular protein . Contribution of TRAIL to betaAP neurotoxicity is demonstrated by data showing that TRAIL-neutralizing monoclonal antibody protects neuronal SH-SY5Y cells from betaAP neurotoxicity . Moreover, exposure of neuronal SH-SY5Y cells to TRAIL leads to cell death, indicating that this substance per se is endowed with neurotoxic properties . We also found that, similarly to betaAP and TRAIL, activation of the death-domain adaptor protein FADD results in neuronal cell death . Lack of FADD function, by overexpression of its dominant negative, rescued cells from either TRAIL- or betaAP-induced neurotoxicity, supporting the hypothesis that these three molecules share common intracellular pathways . Finally, we found that betaAP strongly activated caspase-8, and the cell-permeable, selective caspase-8 inhibitor z-IETD-FMK prevents both betaAP- and TRAIL-induced neurotoxicity . In view of TRAIL's potency in inducing neuronal death, and its role as mediator of betaAP, it is plausible to hypothesize that TRAIL can be regarded as a molecule that provides substantial contribution to betaAP-dependent cell death, which takes part in the progression of the neurodegenerative process and related chronic inflammatory response.

Toxicol Sci, 2003 Apr, 72(2), 339 - 46 Epub 2003 Mar 07.
Ultrafine carbon black particles enhance respiratory syncytial virus-induced airway reactivity, pulmonary inflammation, and chemokine expression; Lambert AL et al.; Exposure to particulate matter (PM) may exacerbate preexisting respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), bronchitis, and pneumonia . However, few experimental studies have addressed the effects of PM on lower respiratory tract (LRT) viral infection . Respiratory syncytial virus (RSV) is a major etiological agent for LRT infections in infants, the elderly, and the immunocompromised and may lead to chronic wheezing and the development of asthma in children . In this study, we examined the effects of carbon black (CB) on RSV-induced pulmonary inflammation, chemokine and cytokine expression, and airway hyperresponsiveness in a mouse model of RSV . Female BALB/c mice were instilled via the trachea (i.t.) with 1 x 106 plaque forming units (pfu) RSV or with uninfected culture media . On day 3 of infection, mice were i.t . instilled with either 40 micro g ultrafine CB particles or with saline . End points were examined on days 4, 5, 7, and 14 of RSV infection . Viral titer and clearance in the lung were unaffected by CB exposure . Neutrophil numbers were elevated on days 4 and 7, and lymphocyte numbers were higher on days 4 and 14 of infection in CB-exposed, RSV-infected mice . CB exposure also enhanced RSV-induced airway hyperresponsiveness to methacholine, bronchoalveolar lavage (BAL) total protein, and virus-associated chemokines monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1 alpha), and regulated upon activation, normal T cell expressed and secreted (RANTES) . MIP-1 alpha mRNA expression was increased in the alveolar epithelium, where ultrafine particles deposit in the lung . These data demonstrate a synergistic effect of ultrafine CB particles on RSV infection, and suggest a potential mechanism for increased respiratory infections in human populations after PM exposure.

J Microbiol Methods, 2003 May, 53(2), 253 - 62
Phage display for detection of biological threat agents; Petrenko VA et al.; The essential element of any immuno-based detector device is the probe that binds analyte and, as a part of the analytical platform, generates a measurable signal . The present review summarizes the state of the art in development of the probes for detection of the biological threat agents: toxins, bacteria, spores and viruses . Traditionally, the probes are antibodies, which are isolated from sera of immunized animals or culture media of hybridomas . However, the "natural" antibodies may have limited application in the new generation of real-time field detectors and monitoring systems, where stress-resistant and inexpensive long-livers are required . Phage display is a newcomer in the detection area, whose expertise is development of molecular probes for targeting of various biological structures . The probes can be selection from about billion clone libraries of recombinant phages expressing on their surface a vast variety of peptides and proteins, including antigen-binding fragments of antibodies . The selection procedure, like kind of affinity chromatography, allows separating of phage binders, which are propagated in Escherichia coli bacterial cells and purified using inexpensive technology . Although phage display traditionally is focused more on development of medical preparations and studying molecular recognition in biological systems, there are some examples of its successful use for detection, which are presented in the review . To be used as probes for detection, peptides and antibodies identified by phage display are usually chemically synthesized or produced in bacteria . Another interesting aspect is using of the selected phage itself as a probe in detector devices, like sort of substitute antibodies . This idea is illustrated in the review by "detection" of beta-galactosidase from E . coli with "landscape" phage displaying a dense array of peptide binders on the surface.

Anal Biochem, 2003 Mar 15, 314(2), 199 - 205
Reagentless optical sensing of glutamine using a dual-emitting glutamine-binding protein; Tolosa L et al.; Glutamine is a major source of nitrogen and carbon in cell culture media . Thus, glutamine monitoring is important in bioprocess control . Here we report a reagentless fluorescence sensing for glutamine based on the Escherichia coli glutamine-binding protein (GlnBP) that is sensitive in the submicromolar ranges . The S179C variant of GlnBP was labeled at the -SH and N-terminal positions with acrylodan and ruthenium bis-(2,2'-bipyridyl)-1,10-phenanthroline-9-isothiocyanate, respectively . The acrylodan emission is quenched in the presence of glutamine while the ruthenium acts as a nonresponsive long-lived reference . The apparent binding constant, K'(d), of 0.72 microM was calculated from the ratio of emission intensities of acrylodan and ruthenium (I(515)/I(610)) . The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of +/-0.02 microM glutamine . Dual-frequency ratiometric sensing was also demonstrated . Finally, the extraordinary sensitivity of GlnBP allows for dilution of the sample, thereby eliminating the effects of background fluorescence from the culture media.

Andrologia, 2003 Apr, 35(2), 117 - 20
The effect of colloid osmotic pressure in human spermatozoa exposed to hypoosmotic conditions; Correa-Perez JR et al.; The use of a protein source such as serum and albumin had been extensively employed as supplements of culture media for handling and culture of gametes and embryos . Protein molecules behave as colloids in solution and contribute to the osmotic pressure of fluids . The interaction of proteins in solution and spermatozoa needs to be assessed in order to determine their possible role in osmoregulation . The aim of this study was to assess possible osmoregulatory mechanisms of protein supplementation against exposure to hypoosmotic conditions by assessing the sperm's response to those environments . A stock hypoosmotic solution (HOS) was prepared by using a mixture of fructose and sodium citrate and adjusted to an osmotic pressure of 150 mOsm l-1 . Another stock solution was prepared by diluting a preparation of synthetic serum supplement {SSS; 6% (v/v) total protein} with distilled water to obtain an osmotic pressure of 150 mOsm l-1 (hypoosmotic SSS or H-SSS) . Three additional solutions were prepared by mixing the stock HOS and H-SSS solutions in the following proportions (v/v): (i) 75% H-SSS/25% HOS, (ii) 50% H-SSS/50% HOS and (iii) 25% H-SSS/75% HOS . Aliquots of washed spermatozoa from 18 men were diluted 1 : 10 (v/v) with each of the testing solutions and incubated for 60 min . Specimens were assessed on wet mounts for total and specific swelling patterns . Swelling patterns were classified as maximal (>50% tail length swollen) and minimal (<50% tail length swollen) swelling with or without associated sperm motility . The major finding of this study was that increasing the concentration of protein supplementation resulted in a decrease in the proportion of maximal sperm tail swelling patterns and an increase in the proportion of minimal tail swelling patterns . A proportion of spermatozoa which exhibited minimal swelling patterns were still motile in all solutions tested, and the percentage of those spermatozoa increased as the protein supplementation was also increased in the testing solutions . Incorporation of protein supplementation as described in this study delays the effect of sperm swelling in hypoosmotic conditions.

Blood, 2003 Jul 1, 102(1), 180 - 3 Epub 2003 Mar 20.
Lack of the CD8+ cell anti-HIV factor in CD8+ cell granules; Mackewicz CE et al.; In HIV infection, CD8+ cells show cytotoxic and noncytotoxic anti-HIV activity . The latter function is mediated, at least in part, by a secreted antiviral protein, the CD8+ cell antiviral factor (CAF) . Because antiviral effector molecules, such as perforin and granzymes, reside in the exocytic granules of CD8+ T cells, we examined the possibility that granules contain CAF-like activity . CD8+ cells from HIV-infected individuals showing strong CAF-mediated antiviral activity were induced to release their granule constituents into culture media . Within 1 hour of stimulation, high levels of granzyme B (a primary granule constituent) were found in the culture fluids of previously activated CD8+ cells . The same culture fluids contained no or very low amounts of CAF activity, as measured with HIV-infected CD4+ cells . Maximal levels of CAF activity were not observed until 5 or 7 days after stimulation, consistent with typical CAF production kinetics . In addition, extracts of granules purified from antiviral CD8+ cells did not show any CAF activity, whereas the cytoplasmic fraction of these cells showed substantial levels of antiviral activity . These findings suggest that CAF does not reside at appreciable levels in the exocytic granules of antiviral CD8+ T cells.

Reprod Biol Endocrinol . 2003 Feb 11;1(1):16.
Synchronous onset of oestradiol-17beta secretion by Meishan conceptuses; Pickard AR et al.; The response of Meishan conceptuses to an exogenous precursor for oestradiol-17beta biosynthesis was investigated in vitro, to determine whether gestational age or morphological stage of development elicit changes in hormone metabolism . Conceptuses were recovered on days 11, 12, 13 or 15 after the onset of oestrus and cultured for 6 hours at 37 degrees C, in the presence or absence of testosterone . On days 12 and 13 after the onset of oestrus spherical conceptuses were recovered from some gilts, whereas others yielded elongated or filamentous conceptuses . All conceptuses recovered on day 15 after oestrus had elongated . The number of cells per individual conceptus increased from days 11 to 13 after the onset of oestrus (P < 0.001), as did conceptus surface area (P = 0.038) . Supplementing culture media with testosterone, as a substrate for oestrogen biosynthesis, significantly increased conceptus oestradiol-17beta secretion in vitro on days 12, 13 and 15, regardless of whether pre- or post-elongation conceptuses were cultured . However, on day 11 oestradiol-17beta was only detected at significant concentrations in the culture media of four testosterone supplemented conceptuses and only one gilt produced conceptuses capable of secreting oestradiol-17beta in the absence of testosterone . Therefore, the onset of conceptus oestradiol-17beta secretion is apparently limited by the expression of aromatase enzymes that are activated synchronously, irrespective of the stage of morphological development, within Meishan litters . Once established, Meishan conceptus oestradiol-17beta secretion in vitro is increased in the presence of exogenous testosterone.

J Proteome Res, 2002 Jul-Aug, 1(4), 337 - 43
Array-based multiplexed screening and quantitation of human cytokines and chemokines; Wang CC et al.; HydroGel-coated slide is a porous substrate based on a polymer matrix that provides a three-dimensional hydrophilic environment similar to free solution suitable for biomolecular interactions . This substrate has been used to develop fluorescence-based multiplexed cytokine immunoassays . Forty-three monoclonal antibodies (mAb) of cytokines and chemokines were printed at a volume of 350 pL per spot using a Packard BioChip Arrayer . For each probe, four replicates were printed at a pitch of 500 microm in the layout of a 13 x 16 pattern on a 12 x 12 mm2 HydroGel pad . Cytokines and chemokines that are captured by the arrayed mAbs are detected by using another biotinylated mAb, following by the addition of a Texas Red-conjugated streptavidin . The fluorescent images of arrays were recorded using a Packard ScanArray 5000 confocal slide scanner and quantitated using Packard QuantArray software . Experiments demonstrated that 43 cytokines and chemokines could be simultaneously screened and quantitated in conditioned culture media, cell lysates, and human plasma . Using this chip, we have examined cytokine expression in breast cancer cells and identified the chemokines associated with human cervical cancers.

J Proteome Res, 2002 Mar-Apr, 1(2), 111 - 4
Near-field optical analysis of living cells in vitro; Sommer AP et al.; Near-field optical analysis (NOA) provides morphological nanoscale mappings of living cells in liquid cell culture media and nondestructive insight into cell functionality . Here we show for the first time the performance of NOA in imaging living cells . Unlabeled human endothelial cells attached to polished titanium disks were analyzed with hydrophobically coated optical biosensors mounted to a near-field scanning optical microscope (NSOM) . Biosensors and titanium substrates could be simply implemented in standard NSOM and high-throughput NOA.

J Chromatogr A, 2003 Mar 7, 989(1), 155 - 63
Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use; Brorson K et al.; A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers of chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time . To define chromatography performance attributes that best predict retrovirus clearance during extended re-use of protein A media, small-scale protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (6 M urea, 6 M guanidine, 100 mM NaOH or 500 mM NaOH) . Load, flow-through and eluate samples were taken periodically and assayed for reverse transcriptase (RT, an enzyme component of retroviruses) activity, bovine IgG (a component of the culture media), genomic DNA, leached protein A, and mouse IgG . Under all cleaning conditions tested, the log,10 reduction value (LRV) of RT activity did not decrease and impurity co-elution did not increase during the 150 to 460 purification/cleaning cycles . In the two studies in which the columns were cleaned with NaOH, the chromatography performance attribute that best predicted the column media lifespan was column capacity, as measured by antibody (Ab) step yield and breakthrough . In both studies, Ab capture decayed in a biphasic manner starting at cycle 200 (100 mM NaOH) or cycle 50 (500 mM NaOH) . For media cycled 300+ times using 6 M urea or 6 M guanidine cleaning buffers, column performance, including RT activity LRV, was more stable, although small upward trends in Ab breakthrough were evident . In summary, our studies identify Ab step yield and breakthrough as performance attributes that decay prior to retrovirus LRV when protein A media is multiply-cycled . Thus, we propose that virus removal validation studies should be performed on new media only and these attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media.

Adv Dent Res, 2001 Aug, 15, 72 - 5
The effects of high levels of glucose and insulin on type I collagen synthesis in mature human odontoblasts and pulp tissue in vitro; Valikangas L et al.; High levels of dietary sucrose affect the metabolism of the pulp-dentin complex and enhance the caries process in dentin . The high-sucrose diet reduces dentin formation in young rats (Tjaderhane et al., 1994; Hietala and Larmas, 1995; Tjaderhane, 1996) and in pups of rat dams fed high-sucrose diet during lactation (Pekkala et al., 2000a) . However, the mechanisms behind the effects are unknown . A direct effect of elevated blood glucose or an indirect effect via insulin has been suggested . We investigated the effects of high glucose and insulin on type I collagen synthesis in human odontoblasts and pulp tissue in vitro, using an organ culture method for functional post-mitotic odontoblasts . Odontoblasts and pulp tissue were cultured separately for 10 days in DMEM with 15% FBS containing additional glucose (G) (4.45 g/L) or insulin (I) (0.6 microgram/mL) or both together (GI) . We evaluated type I collagen synthesis with RIA, measuring the level of N-terminal propeptide of type I collagen (PINP) secreted into the culture media . PINP secretion decreased in odontoblasts and pulp tissue in G and GI groups when compared with the control and insulin samples (p = 0.001 in both groups in the pulp samples) . Insulin alone did not affect PINP secretion distinctly . The results indicate that high levels of glucose, but not insulin, directly down-regulate the type I collagen synthesis in young, differentiated human odontoblasts and pulp tissue . Insulin does not affect the inhibitory effect of high sucrose . These in vitro findings indicate that the high-sucrose diet may alter odontoblast function independently of insulin.

Adv Dent Res, 2001 Aug, 15, 55 - 8
Human odontoblast culture method: the expression of collagen and matrix metalloproteinases (MMPs); Tjaderhane L et al.; Studies on mature human odontoblasts have suffered for the lack of in vitro models . We recently introduced a human odontoblast and pulp tissue organ culture method, in which the odontoblasts are cultured in the pulp chamber after removal of the pulp tissue, and the pulp tissue can be cultured separately (Tjaderhane et al., 1998a) . With this method, we have studied the effects of growth factors on the expression of collagen and extracellular matrix (ECM)-degrading enzymes, matrix metalloproteinases (MMPs), in mature human odontoblasts . TGF-beta 1 was selected because of its ability to regulate the response of the dentin-pulp complex to external irritation . The effect of TGF-beta 1 (10 ng/mL) on pro alpha 1(I) collagen mRNA was analyzed by quantitative PCR, and type I procollagen propeptide (PINP) was analyzed from conditioned culture media with RIA . Odontoblast media were also assayed for respective type III procollagen propeptide (PIIINP) . TGF-beta had a negligible effect on collagen mRNA expression or protein synthesis, indicating that TGF-beta alone does not markedly induce dentin matrix formation per se in the human dentin-pulp complex (Palosaari et al., 2001) . However, TGF-beta 1 seems to regulate MMP expression in mature human odontoblasts differentially . A strong down-regulation of MMP-8 (Palosaari et al., 2000), a modest down-regulation of MMP-20 (Tjaderhane et al., 2000), and considerable up-regulation of MMP-9, with no apparent effect on MMP-2 expression (Tjaderhane et al., 1998b), indicate that growth factors may affect the matrix synthesis by controlling the expression and activity of MMPs instead of collagen synthesis . The altered expression of MMPs may result in altered ECM formation, which in turn may contribute to the formation of atubular reparative dentin.

Microbiol Immunol, 2003, 47(1), 105 - 7
High molecular weight factor in FCS inhibits Helicobacter pylori VacA-binding to its receptor, RPTPbeta, on AZ-521; Kimura T et al.; VacA, a secretory product of Helicobacter pylori, binds to its cell surface receptor, receptor tyrosine phosphatase (RPTP) beta, leading to cytoplasmic vacuolization of gastric epithelial AZ-521 cells . VacA binding to the cell surface and VacA-dependent vacuolization were inhibited by cell culture media containing fetal calf serum (FCS) . The high molecular weight fraction of FCS isolated by Superose 12 gel filtration chromatography inhibited VacA binding, whereas only weak effects were observed with other fractions . These data show that the high molecular weight fraction of FCS inhibits VacA action though its ability to block toxin binding to its receptor, RPTPbeta, on AZ-521 cells.

Kidney Int, 2003 Feb, 63(2), 722 - 31
High glucose levels inhibit focal adhesion kinase-mediated wound healing of rat peritoneal mesothelial cells; Tamura M et al.; BACKGROUND: The peritoneum is progressively denuded of its mesothelial cell monolayer in patients on continuous ambulatory peritoneal dialysis (CAPD) . These alterations of the mesothelium cause membrane dysfunction and progressive peritoneal fibrosis . Integrins regulate cell motility and play an important role in wound healing . We investigated the effects of high glucose on the regeneration process of the peritoneal mesothelial cell monolayer using cultured rat peritoneal mesothelial cells (RPMC) . METHODS: The effects of glucose or mannitol on the regeneration of RPMC and formation of focal adhesions were examined by in vitro wound healing assay and immunocytochemistry, respectively . Activities of focal adhesion kinase (FAK) and its downstream p130Cas were examined by Western blotting . Effects of wild-type and dominant-negative FAK on RPMC migration were examined by a transient transfection assay . RESULTS: Cell migration over fibronectin (FN) was clearly inhibited in culture media containing high glucose (28 to 140 mmol/L) . RPMC formed focal adhesions on FN in the presence of a regular glucose concentration (5.6 mmol/L); however, tyrosine phosphorylation of FAK and p130Cas and formation of focal adhesions observed by FAK and vinculin staining were substantially inhibited by high glucose . Mannitol also induced significant inhibitory effects, but these were milder than those of glucose . Transfection of dominant-negative FAK inhibited cell migration in a regular glucose concentration, whereas overexpression of wild-type FAK abrogated glucose-induced inhibition of cell migration . CONCLUSIONS: Our results demonstrate that high glucose concentrations as well as high osmolarity inhibit FAK-mediated migration of mesothelial cells, and suggest that dialysates containing high glucose concentrations may cause peritoneal damage by inhibiting wound healing of the mesothelial cell monolayer.

Hypertens Res, 2003 Feb, 26 Suppl, S41 - 4
Decreased expression of adrenomedullin during adipocyte-differentiation of 3T3-L1 cells; Li Y et al.; Adrenomedullin (AM) is a potent vasodilator peptide which has an inhibitory action on insulin secretion . Resistin is a novel peptide specifically secreted from adipocytes, and implicated in insulin resistance . We studied the expression of AM and resistin in 3T3-L1 adipocytes and preadipocytes by Northern blot analysis and radioimmunoassay . Immunoreactive-AM was detected in the culture media of 3T3-L1 preadipocytes and adipocytes, with higher concentrations found in preadipocytes . Northern blot analysis showed that AM mRNA was expressed in 3T3-L1 preadipocytes but was undetectable in adipocytes . In contrast, resistin mRNA was expressed in 3T3-L1 adipocytes, whereas it was not detected in 3T3-L1 preadipocytes . The present study thus showed that AM expression was decreased, and resistin expression increased, during adipocyte-differentiation of 3T3-L1 cells.

Folia Biol (Praha), 2003, 49(1), 33 - 9
Two dynamic morphotypes of sarcoma cells, asymmetric stellate and triangle with leading lamella, are related to malignancy; Pokorna E et al.; A notion of the dynamic morphotype was developed as a conjunction between cell shape and migration . This enabled the investigation of the relationship between malignancy and patterns of dynamic morphology in neoplastic cells in vitro . Time-lapse cinemicroscopy was used to analyse the cell behaviour of three rat neoplastic cell lines (K2, T15, and A8), differing in metastatic potential, that were instrumental in revealing a coincidence between high migratory activity and appearance of the 3D structure of actin cables in high-malignant A8 cells (Pokorna et al., 1994) . A set of criteria was established for visual classification of cell morphology . Matching the pattern of cell morphology with locomotory activity led to identification of four dynamic morphotypes . Cell speed was determined by tracking and the dynamic morphotypes assigned by the operator . All the three cell populations were studied for incidence of the dynamic morphotypes in culture media differing in pH: 6.6 simulating acid extracellular condition in tumours, physiological 7.4, and alkaline 8.2 . The results showed that acid pH stimulated motile activity in the intermediate-malignant T15 and most malignant A8 cells . The T15 and A8 cells also manifested a prolonged continuation of fast locomotion in the early G1 phase and displayed a prevalence of two fast moving dynamic morphotypes: asymmetric stellate and triangle with leading lamella.

Environ Toxicol Chem, 2003 Mar, 22(3), 576 - 85
Comparison of the response of three microalgae species exposed to elutriates of estuarine sediments based on growth and chemical speciation; Mucha AP et al.; The elutriate sediment toxicity test (ESTT) provides a measure of the amount of a substance that is exchanged between the sediment and the aqueous phase during resuspension processes such as floods or dredging operations . This study used ESTT with two complementary aims: a comparison of the elutriates of two estuarine sediments (anaerobic muddy {A} and aerobic sandy {B}) in terms of toxicity and a comparison of the response of three different microalgae (Emiliania huxleyi (coccolithophore), Dunaliella minuta (green alga), and Phaeodactylum tricornutum (diatom)) to each elutriate in terms of growth, heavy metals uptake, and organic ligands release or uptake in order to find eventual differences of sensitivity . The interpretation of the results was based on chemical speciation in the culture media . Both elutriates, particularly A, were much richer than seawater (control medium) in some heavy metals and organic ligands able to bind strongly heavy metals . Elutriate A slightly inhibited P . tricornutum growth but stimulated growth of E . huxleyi and D . minuta . Elutriate B stimulated the growth of the three algal species . Therefore, the diatom behaved differently from both the coccolithophore and the green alga . Strong complexation of trace metals by organic ligands could be the cause of absence of the metallic toxicity of the elutriates . Growth inhibition of P . tricornutum in elutriate A could be caused by ammonia-N and/or organic compounds . The concentration of the organic ligands decreased markedly (about 75%) in both elutriates after 10 d of incubation in contrast to the control culture, where their concentration increased about 50% because of exudation . This phenomenon was interpreted to result from ligand uptake by the algae, free or as metal complexes . This work demonstrated that beside the evaluation of toxicity of free heavy metals to alga species, the organic ligands must not be ignored . Depending on the amount of ligand present, the toxicity can be reduced (sequestration) or enhanced (better availability through uptake of metal-ligand complexes) . Since the applied ESTT is a standard procedure (U.S . Environmental Protection Agency) for the evaluation of dredged material proposed for ocean disposal, it is necessary to discuss results obtained during toxicity tests with such elutriates in detail.

J Appl Physiol, 2003 Jul, 95(1), 448 - 53; discussion 435 Epub 2003 Mar 07.
Adaptation to chronic length change in explanted airway smooth muscle; Naghshin J et al.; It has been shown that airway smooth muscle in vitro is able to maintain active force over a large length range by adaptation in the absence of periodic stimulations at 4 degrees C (Wang L, Pare PD, and Seow CY . J Appl Physiol 90: 734-740, 2001) . In this study, we show that such adaptation also takes place at body temperature and that long-term adaptation results in irreversible functional change in the muscle that could lead to airway hyperresponsiveness . Rabbit tracheal muscle explants were passively maintained at shortened and in situ length for 3 and 7-8 days in culture media; the length-tension relationship was then examined . The length associated with maximal force generation decreased by 10.5 +/- 3.8% (SE) after 3 days and 37.7 +/- 8.5% after 7 or 8 days of passive shortening . At day 3, the left shift in the length-tension curve due to adaptation at short lengths was reversible by readapting the muscle at a longer length . The shift was, however, not completely reversible after 7 days . The results suggest that long-term adaptation of airway smooth muscle could lead to increased muscle stiffness and force-generating ability at short lengths . Under in vivo condition, this could translate into resistance to stretch-induced relaxation and excessive airway narrowing.

Hypertension, 2003 Mar, 41(3 Pt 2), 715 - 9 Epub 2002 Dec 30.
Heme oxygenase attenuates angiotensin II-mediated increase in cyclooxygenase-2 activity in human femoral endothelial cells; Li Volti G et al.; Heme oxygenase (HO) regulates cellular heme levels and catalyzes the formation of bilirubin and carbon monoxide . We hypothesize that the status of the endothelial HO system influences the angiotensin (Ang) II-induced increase in the endothelial production of prostaglandin I2 (PGI2) (measured as 6-keto-PGF1alpha) and prostaglandin E2 (PGE2), eicosanoids that modulate the vascular actions of Ang II . In the present study, we determined the effect of interventions that suppress HO activity or induce HO-1 gene expression on Ang II-mediated increase in 6-keto-PGF1alpha and PGE2 in cultures of human femoral artery endothelial cells . Incubation of endothelial cells with Ang II (100 ng/mL) for 24 hours increased the levels of both 6-keto-PGF1alpha and PGE2 in the culture media . This effect of Ang II on prostaglandin production by endothelial cells was attenuated in cells treated with SnCl2 (10 micromol/L), an inducer of HO-1, but was magnified in cells treated with the HO inhibitor ZnDPP or heme . Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated heme and Ang II-induced prostaglandin synthesis . Of note, prostaglandin synthesis by lysates of endothelial cells stimulated with heme or Ang II appear to involve COX-2, because it was blunted by NS-398, which is presumed to inhibit COX-2 specifically . These results indicate that overexpression of the HO system exerts an inhibitory influence on Ang II-induced synthesis of prostaglandins by endothelial cells.

Fertil Steril, 2003 Mar, 79 Suppl 1, 821 - 7
Interleukin-13 and tumor necrosis factor-beta differentially regulate the production of cytokines by cultured human endometrial stromal cells; Nasu K et al.; OBJECTIVE: To evaluate the effects of interleukin (IL)-13, a T-helper (Th)2 cytokine, and tumor necrosis factor (TNF)-beta, a Th1 cytokine, on the production of IL-6 family cytokines and chemokines by endometrial stromal cells (ESC) . DESIGN: The effects of IL-13 and TNF-beta, on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene alpha (GROalpha), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, T-cell expressed and secreted (RANTES), and eotaxin were investigated . SETTING: Research laboratory at a medical university . PATIENT(S): Thirteen endometrial specimens in the late proliferative phase were used . INTERVENTION(S): The ESC were incubated for 24 hours with recombinant human IL-13 and recombinant human TNF-beta . MAIN OUTCOME MEASURE(S): The concentration of IL-6, IL-11, LIF, IL-8, GROalpha, MCP-1, RANTES, and eotaxin in the culture media was measured using ELISA . RESULT(S): The increase in levels of IL-6, IL-8, MCP-1, and eotaxin in the culture media of ESC paralleled the addition of increasing amounts of IL-13 and TNF-beta, whereas the levels of IL-11 and LIF were decreased with increasing amounts of IL-13, but were increased with increasing amounts of TNF-beta . Tumor necrosis factor-beta enhanced the production of GROalpha and RANTES in dose-dependent manner; however, IL-13 did not affect the expression of GROalpha or RANTES . CONCLUSION(S): These results suggest that IL-13 and TNF-beta secreted in the cyclic endometrial tissue and in the decidua may differentially regulate the production of IL-6 family cytokines and chemokines by ESC . The controlled expression of these cytokines in the endometrium may contribute to the modulation of the immune reaction during the menstrual cycle and in early pregnancy by the regulation of leukocyte trafficking and functions.

Fertil Steril, 2003 Mar, 79 Suppl 1, 802 - 7
Cumulus cells reduce the spermatozoa-zona binding inhibitory activity of human follicular fluid; Hong SJ et al.; OBJECTIVE: To investigate the effects of human follicular fluid cultured with cumulus cells to inhibit the binding of spermatozoa to the zona pellucida of oocytes . DESIGN: Controlled experimental laboratory study . SETTING: University gynecology unit . PATIENT(S): Women undergoing assisted reproduction program and men visiting the subfertility clinics . INTERVENTION(S): Culture medium and human follicular fluid were used to culture cumulus cells in vitro for specified time periods . MAIN OUTCOME MEASURE(S): Zona binding capacity and motility of spermatozoa after incubation with cumulus cells treated culture medium or human follicular fluid . RESULT(S): Compared with the control medium, spent culture media after culturing cumulus cells for 3, 5, and 7 hours did not affect the motility and zona binding capacity of the treated spermatozoa . Significantly more spermatozoa treated with human follicular fluid that had been preincubated with cumulus cells for 5 and 7 hours bound onto hemizona in hemizona binding assay when compared with those preincubated in human follicular fluid without cumulus treatment . The hemizona index increased with the increase in the duration of cumulus cell treatment . Human follicular fluid with or without cumulus cells maintained sperm motility to similar extent for 3 hours . CONCLUSION(S): Cumulus cells reduced the inhibitory effect of human follicular fluid on spermatozoa-zona binding in vitro in a time-dependent manner.

Leuk Res, 2003 May, 27(5), 455 - 64
In vitro culture of human acute lymphoblastic leukemia (ALL) cells in serum-free media; a comparison of native ALL blasts, ALL cell lines and virus-transformed B cell lines; Bruserud O et al.; The aim of this study was to standardize in vitro culture conditions for human acute lymphoblastic leukemia (ALL) cells . The cells were cultured in medium containing 10% fetal calf serum (FCS) and in the four serum-free media X-vivo 10, X-vivo 15, X-vivo 20 and Stem Span . Native ALL blasts could proliferate in all four serum-free media, but the strongest responses were usually observed with Stem Span . Native leukemia blasts were also cultured in the presence of various single cytokines or cytokine combinations . The highest proliferation was usually observed in the presence of Flt3-Ligand (Flt3-L) when single cytokines were examined, and these responses could be further increased especially by combining Flt3-L with interleukin 3 (IL3), IL7 or stem cell factor (SCF) . Proliferation could also be increased when ALL blasts were cultured in the presence of two commercially available fibroblast cell lines (Hs27 and HFL1) . Based on these results we suggest that in vitro culture conditions for native human ALL blasts can be standardized by using serum-free culture media supplemented with exogenous Flt3-L+IL3+SCF, and the use of accessory cells can also be standardized by using well-characterized fibroblast cell lines . Detectable ALL blast proliferation can then be observed for most patients . Our experimental model can thereby be used for in vitro evaluation of possible antileukemic treatment strategies, and it will then allow comparison of experimental results between different studies.

Compend Contin Educ Dent, 2003 Jan, 24(1), 68 - 72, 74
Intentional replantation of endodontically treated teeth: an update; Wolcott J et al.; The IR technique is a clinically successful procedure, so long as the following conditions, as outlined by Niemczyk, are met: 1) Avoid any crushing or scraping contact with the root surface or socket; 2) Root surface must be continually hydrated with tissue culture media (e.g., HBSS); 3) Tooth should be splinted, if indicated; and 4) Soft diet and hygiene instructions must be implemented and reinforced . The IR technique should not be considered a procedure of last resort . Rather, it should be used in situations where conventional apical surgery is difficult or places the patient at risk . The IR technique expands potential treatment alternatives and allows the patient to successfully retain his or her own tooth following treatment.

Vet J, 2003 Jan, 165(1), 78 - 83
Escherichia coli in the rumen and colon of slaughter cattle, with particular reference to E . coli O157; Laven RA et al.; The distribution of Escherichia coli O157 and of total E . coli was surveyed in the digestive tract of cattle under 30 months of age, slaughtered between August 1999 and May 2000 in three abattoirs in southern England . Samples were taken from the dorsal and ventral rumen wall, the rumen contents, the colon wall and colon contents, and from faeces or caudal rectal contents . Gut wall samples were processed by vortex-mixer to release loosely adherent bacteria, and by Stomacher to release firmly attached bacteria . E . coli O157 was detected by immunomagnetic separation followed by growth on selective culture media . The numbers of E . coli were higher in the colon than the rumen, and most were located in the digesta phase, rather than associated with the gut wall . The number of E . coli found in the gut and in faeces decreased during the winter months . E . coli O157 was detected more frequently in the colon than in the rumen, but the majority of detections(7/8) were in samples of rumen wall.

Reprod Fertil Dev, 2002, 14(7-8), 443 - 51
The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro; Figueiredo F et al.; The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts . Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium . Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further . The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium . Development to blastocyst was similar, regardless of the macromolecule used . The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls . Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells . It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development . Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte-macrophage colony-stimulating factor.

J Agric Food Chem, 2003 Mar 12, 51(6), 1718 - 23
Antiproliferative activity of apples is not due to phenolic-induced hydrogen peroxide formation; Liu RH et al.; Anticancer compound screening of natural products using tumor cell lines has been commonly used to identify anticancer drugs . Two highly significant anticancer drugs, paclitaxel (Taxol) and camptothecin, were discovered using tumor cell lines by the U.S . National Cancer Institute (NCI) screening program of plants . It has been recently reported that the inhibition of cancer cell proliferation by fruit extracts was indirectly caused by phenolic-induced H(2)O(2) production in the cell culture media, suggesting that many previously reported effects of flavonoids and phenolic compounds on cultured cells might be from an artifact of H(2)O(2)-induced oxidative stress . The objective of the present study was to determine if apple extracts induced H(2)O(2) formation in common cell culture media and to investigate if the antiproliferative activity of apple extracts was due to phenolic-induced H(2)O(2) formation . It is reported here that apple extracts did not induce H(2)O(2) formation in WME, DMEM, or DMEM/Ham F12 media with the cell culture conditions tested . These same extracts inhibited proliferation of HepG(2) and Caco-2 cells . Therefore, antiproliferative activity of apple extracts was not due to the phenolic-induced H(2)O(2) production in cell culture media . In addition, H(2)O(2) added to the culture medium at 100 microM did not cause inhibition of cell proliferation in either HepG(2) liver cancer cells or Caco-2 colon cancer cells in vitro.

Int J Pharm, 2003 Mar 18, 254(1), 27 - 31
Formulation and stability of surface-tethered DNA-gold-dendron nanoparticles; Hussain N et al.; The formulation of plasmid DNA on 100 nm gold nanoparticles surface-tethered via cationic dendrons, and the behaviour of the complex in cell culture media, is described in this communication . Adsorption of dendrons onto gold nanoparticles in water resulted in the generation of positively charged nanoparticles with a corresponding small increase in particle size . Addition of plasmid DNA did not markedly reduce the surface potential but resulted in a approximately 10-20% increase in hydrodynamic diameter . More dramatic effects were seen in the presence of cell culture media that, overall, drastically increased the apparent size of the gold-dendron-DNA nanoparticles and reduced the surface potential of the colloids, the presence of serum components partially ameliorating these effects possibly due to steric stabilisation . Release of the surface-tethered DNA was reduced in cell culture media compared to water . This reduced detachment of DNA coupled with the flocculation of the carrier which would likely inhibit endocytosis, demonstrates the importance of testing drug delivery systems with relevant physiologically based fluids prior to their use in vivo studies .

Diagn Microbiol Infect Dis, 2003 Feb, 45(2), 127 - 30
Effectiveness of peptone-yeast extract (P-Y) medium in the cultivation and isolation of Entamoeba histolytica/Entamoeba dispar in Turkish patients; Dagci H et al.; Amebiasis is a common protozoan infection worldwide, causing serious health problems in both children and adults . Today, almost 10% of the world population is infected with Entamoeba histolytica/Entamoeba dispar . The aims of this study were both the comparison of the reproduction rates and densities of E . histolytica/E . dispar in Robinson, Dobell-Laidlaw and P-Y culture media and isolation of E . histolytica/E . dispar from stool samples in Peptone-Yeast (P-Y) medium . Trophozoites and cysts of E . histolytica/E . dispar, maintained in Robinson medium, and stool samples of patients with amebiasis were inoculated into P-Y, Robinson and Dobell-Laidlaw culture media . Reproduction rates reached their peak levels 48 h after the inoculation in all culture media . Reproduction rates in P-Y and Robinson media were found similar; however, they were higher than the reproduction rate in Dobell-Laidlaw medium (p < 0.01); there was no statistically significant difference between the reproduction rates of P-Y and Robinson media (p > 0.05) . Twelve isolates from 12 patients were cultivated in P-Y medium and checked for reproduction everyday for 7 days . Twelve of the 12 (100%) isolates were cultivated in P-Y medium, indicating that the P-Y was an effective medium for the isolation of E . histolytica/E . dispar in stool samples . According to these results, P-Y medium could be preferred in immunologic, serologic and molecular studies and, thus the definitive diagnosis of amebiasis due to its low cost and simple formula.

Reproduction, 2003 Mar, 125(3), 437 - 46
Presence of LH receptor mRNA in granulosa cells as a potential marker of oocyte developmental competence and characterization of the bovine splicing isoforms; Robert C et al.; As the expression of the LH receptor (LH-R) in granulosa cells is thought to be associated with later stages of folliculogenesis, this study was undertaken to evaluate the presence of LH-R mRNA as a suitable marker for developmental competence of oocytes . Granulosa cells and cumulus-oocyte complexes (COCs) were recovered from cows that had received ovarian stimulation . The COCs were subjected to embryo production procedures in vitro to assess the embryonic potential of the oocyte, and the corresponding granulosa cells were used to evaluate the presence of LH-R mRNA by RT-PCR . The presence of LH-R transcripts in granulosa cells is not a key characteristic of a follicle bearing a competent oocyte, although a higher proportion of oocytes reach the blastocyst stage when LH-R mRNA is detected in the granulosa cells . Different LH-R isoforms were cloned and sequence discrepancies among six of the isoforms enabled the design of specific oligonucleotides to study the presence of the isoforms in different follicular cells . All LH-R transcripts studied and the 80 kDa protein product corresponding to the full length receptor were found in granulosa cells of small (< 4 mm) and large (> 5 mm) follicles . When the granulosa cells were cultured, the transcripts were downregulated by the culture conditions; downregulation was more acute in granulosa cells from small follicles . The addition of LH to the culture media enhanced LH-R mRNA downregulation . The presence of several LH-R transcript isoforms was tissue specific and in the theca cells LH-R mRNA was restricted mainly to cells from larger follicles . This finding indicates that the expression and the splicing of LH-R mRNA are regulated in a cell-specific and follicular size-specific manner.

J Virol, 2003 Mar, 77(6), 3371 - 83
Induction of apoptosis by paramyxovirus simian virus 5 lacking a small hydrophobic gene; Lin Y et al.; Simian virus 5 (SV5) is a member of the paramyxovirus family, which includes emerging viruses such as Hendra virus and Nipah virus as well as many important human and animal pathogens that have been known for years . SV5 encodes eight known viral proteins, including a small hydrophobic integral membrane protein (SH) of 44 amino acids . SV5 without the SH gene (rSV5deltaSH) is viable, and growth of rSV5deltaSH in tissue culture cells and viral protein and mRNA production in rSV5deltaSH-infected cells are indistinguishable from those of the wild-type SV5 virus . However, rSV5deltaSH causes increased cytopathic effect (CPE) and apoptosis in MDBK cells and is attenuated in vivo, suggesting the SH protein plays an important role in SV5 pathogenesis . How rSV5deltaSH induces apoptosis in infected cells has been examined in this report . Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, was detected in culture media of rSV5deltaSH-infected cells . Apoptosis induced by rSV5deltaSH was inhibited by neutralizing antibodies against TNF-alpha and TNF-alpha receptor 1 (TNF-R1), suggesting that TNF-alpha played an essential role in rSV5deltaSH-induced apoptosis in a TNF-R1-dependent manner . Examination of important proteins in the TNF-alpha signaling pathway showed that p65, a major NF-kappaB subunit whose activation can lead to transcription of TNF-alpha, was first translocated to the nucleus and was capable of binding to DNA and then was targeted for degradation in rSV5deltaSH-infected cells while expression levels of TNF-R1 remained relatively constant . Thus, rSV5deltaSH induced cell death by activating TNF-alpha expression, possibly through activation of the NF-kappaB subunit p65 and then targeting p65 for degradation, leading to apoptosis.

J Immunol Methods, 2003 Mar 1, 274(1-2), 199 - 207
Studies on interaction between hTNF-alpha and its two receptors with expressed hsTR55-preS1/hsTR75-preS1 fusion soluble receptors; Fang J et al.; The gene encoding the N-terminal 2-50 amino acids of HBsAg-preS1 was amplified by PCR and fused to the 3'-end of two human soluble TNF receptor genes to form the hsTR55-preS1/hsTR75-preS1 fusion genes . The recombinant bicistronic expression vectors were further constructed, which contained one of the human soluble TNF receptor fusion genes and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), followed by the neomycin phosphotransferases as the selectable marker . BHK-21 cells transfected with those vectors by electroporation were selected with G-418, and the positive colonies expressing the protein of interest were obtained . All of the culture media of those transfects could fairly neutralize hTNFalpha-induced cytotoxicity to L929 cells . The expression of hsTR55-preS1/hsTR75-preS1 in those cells has been further demonstrated by RT-PCR and indirect ELISA at RNA transcription and protein translation levels . Their K(d) (dissociation constant) value has also been assayed with BIOSENSOR method . The results showed that the fused HBsAg-preS1 peptide did not affect the dissociation constant of hsTR55 or hsTR75 with hTNFalpha and its muteins . Thus, a novel nonradioactive ELISA method was developed for studies on interaction between hTNFalpha and its two receptors using those expressed fusion receptors.

Biol Reprod, 2003 Jul, 69(1), 48 - 56 Epub 2003 Feb 05.
Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei; Gao S et al.; Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming) . Failure to silence the donor cell program could lead to altered embryonic phenotypes . Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos . Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos . Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface . Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake . These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development . As a result, cloned embryos initially exhibit many somatic cell-like characteristics . Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference . We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.

Breast Cancer Res Treat, 2003 Jan, 77(2), 125 - 31
Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells; Gebauer G et al.; Cytotoxic activity of drug conjugates of human chorionic gonadotropin (hCG) and doxorubicin alone was investigated compared to doxorubicin in breast cancer cells with and without expression of hCG receptors . Expression of hCG receptor was determined in MCF-7 and MB231 breast cancer cell line using a multiplex nested rt-PCR approach . The entire sequence of mRNA encoding for hCG receptor was detected in MCF-7 but not in MB231 breast cancer cell line . Cytostatic effect of doxorubicin-hCG conjugates was investigated in these cell lines in comparison to unconjugated doxorubicin . The number of viable cells was determined after 24, 48, 72, 96, and 120h . To exclude non-specific uptake of the carrier hCG from the culture media, a similar experiment was performed with albumin-doxorubicin conjugates . The number of viable cells decreased in a concentration depending manner after doxorubicin and hCG-doxorubicin conjugate treatment . However, the cytotoxic effect of hCG-doxorubicin conjugate was 10-fold increased compared to unconjugated doxorubin in hCG-receptor positive MCF-7 but not in hCG-receptor negative MB231 cells . Albumin-doxorubicin conjugates showed no increased toxicity compared to doxorubicin . We conclude that the cytotoxic effect of hCG-doxorubicin conjugates is mediated specifically via the hCG receptor . By using hCG conjugates, the development of more selective cytostatics can be achieved.

Zhejiang Da Xue Xue Bao Yi Xue Ban, 2002 Aug, 31(6), 424 - 428
{Signal transduction pathways induced by nitric oxide in rat hepatocytes}; Chen Y et al.; OBJECTIVE: To study signal transduction pathways in cultured rat hepatocytes in the high nitric oxide (NO) environment of hepatitis . METHODS: NO levels were assessed by measurement of its stable oxidative products nitrite (NO2(-)) and nitrate (NO3(-)) using the Griess method with or without thiols (GSH or L-Cys) . Rat hepatocytes were incubated with Sodium Nitroprusside (SNP) to produce a high NO environment and the intracellular cGMP and s-nitrosoglutathione (GSNO) in the culture media were measured using radioimmunoassay or with the MTT assay absorbed at 334nm respectively . RESULTS: After incubation of 1.543 mmol/L SNP for 30 minutes 0.63+/-0.06 mmol/L and at 25 minutes 0.98+/-0.11 mmol/L of NO was released in containing 25 mmol/L GSH and L-Cys condition . The levels of both cGMP and GSNO were significantly increased (compared with control P<0.05) in a dose related manner . CONCLUSION: Signal transduction of cultured rat hepatocytes in a high NO environment could be a cGMP-dependent as well as a non-cGMP-dependent pathway.

Wei Sheng Yan Jiu, 2002 Aug, 31(4), 223 - 5
{Toxicity effects of manganese on PC12 cells}; Chen J et al.; In order to study the mechanisms of inhibitory effects of manganese on neurocyte . PC12 cells were incubated in culture media with 100, 200 and 500 mmol/L manganese(MnCl2) for 24, 48 and 72 h respectively . MTT test, Trypan blue exclusion test, malondiadehyde(MDA) colorimetry and DNA gel electrophoresis were performed to exam proliferation, lipid peroxidation, and apoptosis of PC12 cells . The results of MTT test reveled that manganese (100-500 mmol/L) could inhibit the proliferation of PC12 cells . DNA gel electrophoresis showed that the apoptosis of the PC12 cells could be induced by manganese (100 mmol/L) . It is concluded that manganese can inhibit proliferation of PC12 cells, the mechanism includes the interference of DNA metabolism, inhibitory of lipid peroxidation, and cell apoptosis.

Rev Argent Microbiol, 2002 Oct-Dec, 34(4), 205 - 12
{Development and application of a method for isolating Escherichia coli O157:H7 in the city of Gualeguaychú}; Tanaro JD et al.; Culture media, reagents, and commercial kits were compared on artificially contaminated food samples . The objective was to find an isolation method for Escherichia coli O157:H7 sensitive, specific and accessible in terms of cost, requirements of equipments and qualification of the analyst . The adopted scheme consisted in a selective enrichment at 42 degrees C during 18 to 24 h, using an appropriate medium, in accordance with the nature of the sample, followed by a step of immunomagnetic separation and simultaneous isolation on a chromogenic agar and MacConkey sorbitol agar with potassium tellurite and cefixime, during 18-24 h at 37 degrees C . The presumptive colonies were confirmed as E . coli O157 by serological and biochemical tests . Secondly, this methodology was applied to food samples, water, bovine gastric content and manure . A total of 410 samples were studied: 279 from meat, 54 milk and dairy products, 6 from vegetables, 27 water samples and 44 bovine gastric content and manure . The frequency of isolation of E . coli O157:H7 was of 3.9% . The phenotypic and genotypic characterization of the isolates was performed . A simple isolation methodology for E . coli O157 was developed, which proved accessible to food laboratories of lower complexity . This methodology allowed the detection of this pathogen in food and environmental samples in Gualeguaychu City . The role of water as vehicle of infection was also established . The strains harbored the same virulence factors as those recovered from human disease.

Neuroreport, 2003 Feb 10, 14(2), 289 - 92
The effect of caspase inhibitors and neurotrophic factors on damaged retinal ganglion cells; Oshitari T et al.; To elucidate the role of caspase inhibitors and neurotrophic factors in retinal ganglion cell (RGC) death and regeneration, we cultured mouse retinal explants in the presence of caspase-1, -3, -8, or -9 inhibitors, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in serum-free culture media . We quantified apoptosis by TUNEL staining in RGCs and assessed the number of regenerating neurites . Apoptosis of RGCs treated with all caspase inhibitors or with neurotrophic factors was significantly reduced and the number of regenerating neurites was significantly greater than controls (p < 0.05) . Our findings indicate that caspase-1, -3, -8, -9 play a critical role in explanted RGC death and may be ideal targets of neuroprotection and regeneration of damaged RGCs.

Tumour Biol, 2002 Sep-Oct, 23(5), 263 - 78
Identification and immunohistochemical characterization of a mucin-like glycoprotein expressed in early stage breast carcinoma; Colpitts TL et al.; In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq Expressed Sequence Tag database . A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types . BS106 mRNA was detected in 90% of the breast tissues examined . The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile . BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media . Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer . BS106 protein was detected in the vast majority of carcinomas (70-100%) and overexpressed in approximately 30% of the 22 specimens analyzed . BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma .

Biochem Biophys Res Commun, 2003 Feb 28, 302(1), 84 - 8
Heparin inhibits SMC growth in the presence of human and fetal bovine serum; Cindhuchao N et al.; Heparin (HP) has antiproliferative as well as anticoagulant properties, but not all HP preparations are equally antiproliferative . A recent report found that HP lost its total antiproliferative activity when fetal bovine serum (FBS) was replaced with human serum (HS) in culture media . This observation led to the investigation of our most potent antiproliferative Upjohn HP preparation effects on bovine pulmonary artery smooth muscle cells (PASMC) and systemic SMC growth stimulated in the presence of either FBS or HS . Bovine PASMC, human PASMC, and bovine aortic SMC were treated with 10 microg/ml Upjohn HP in either 15% FBS or 15% HS and the cell number was determined by a Coulter counter . We found that Upjohn HP significantly inhibited bovine PASMC and systemic SMC proliferation in both HS and FBS . The antiproliferative activity of the above HP preparation in HS may lead to an effective treatment of pulmonary vascular and systemic remodeling.

Anat Embryol (Berl), 2003 Feb, 206(3), 163 - 73 Epub 2003 Jan 24.
Different expression of 25-kDa heat-shock protein (Hsp25) in Meckel's cartilage compared with other cartilages in the mouse; Shimada M et al.; The 25-kDa heat-shock protein (Hsp25) is expressed in the cartilage of the growth plate and suggested to function in chondrocyte differentiation and degeneration . Using immunohistochemistry, we examined the temporal and spatial occurrence of Hsp25 in Meckel's cartilage in embryonic mice mandibles, and in other types of cartilage in both embryonic and adult mice . In adults, Hsp25 immunoreactivity was detected in the hypertrophic chondrocytes located in growth plates of long bones and in non-osteogenic laryngeal and tracheal cartilages . No chondrocytes in the resting or proliferating phase exhibited Hsp25 immunoreactivity . In the embryonic mandibles, resting and proliferating chondrocytes in the anterior and intermediate portions of Meckel's cartilage showed Hsp25 immunoreactivity from the 12th day of gestation (E12) through E15, whereas those in the posterior portion showed little or no immunoreactivity . After E16, the overall Hsp25 immunoreactivity in Meckel's cartilage substantially reduced in intensity, and little or no immunoreactivity was detected in the hypertrophic chondrocytes located in the degenerating portions of Meckel's cartilage . The antisense oligonucleotide for Hsp25 mRNA applied to the culture media of the mandibular explants from E10 embryos caused significant inhibition of the development of the anterior and middle portions of Meckel's cartilage . These results suggested that Hsp25 is essential for the development of Meckel's cartilage and plays different roles in Meckel's cartilage from those in the permanent cartilages and the cartilages undergoing endochondral ossification.

J Pharm Sci, 2003 Mar, 92(3), 594 - 603
Extracellular glucose concentration alters functional activity of the intestinal oligopeptide transporter (PepT-1) in Caco-2 cells; D'Souza VM et al.; The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells . Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol . Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1 . The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR . Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression . In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed . Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier . Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose . In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway .

Nephrol Dial Transplant, 2003 Mar, 18(3), 484 - 90
Suppressive effects of Perilla frutescens on IgA nephropathy in HIGA mice; Makino T et al.; BACKGROUND: Perilla frutescens (perilla) is a herbal medicine used in Japanese traditional Kampo medicine . The present study was conducted to evaluate the anti-nephritic effects of perilla in HIGA mice that spontaneously develop high levels of serum immunoglobulin A (IgA) along with mesangial IgA deposition . METHODS: A perilla decoction and its major active constituent, rosmarinic acid (RsA), were orally administrated to 10-week-old HIGA mice for 16 weeks . At study completion, we measured proteinuria and serum IgA levels and generated histological scores from kidney specimens . In addition, we measured concentrations of IgA in culture media of intestinal Peyer's patch cells and spleen cells obtained from the HIGA mice . RESULTS: Perilla suppressed proteinuria, proliferation of glomerular cells, serum levels of IgA, glomerular IgA and IgG depositions in HIGA mice . Cultured Peyer's patch cells and spleen cells from perilla-treated mice produced significantly less IgA than controls . Rosmarinic acid, by itself, suppressed serum IgA levels and glomerular IgA deposition in HIGA mice . Cultured spleen cells from RsA-treated mice produced less IgA than controls . CONCLUSIONS: The perilla decoction may suppress IgA nephropathy, in part, through modulation of the intestinal mucosal immune system . These effects were caused by RsA acting synergistically with other constituents.

J Biomed Mater Res A, 2003 Mar 1, 64(3), 583 - 90
Porous polymer scaffolds surface-modified with arginine-glycine-aspartic acid enhance bone cell attachment and differentiation in vitro; Hu Y et al.; This study was designed to determine if the surface modification of porous poly(lactic acid) (PLA) scaffolds would enhance osteogenic precursor cell (OPC) attachment, growth, and differentiation . A covalently grafted amino group (-NH(2)), poly(L-lysine) (PLL), and the peptide arginine-glycine-aspartic acid (RGD) were selected for the evaluation . The hypothesis was that surface modification would have a positive impact on cell-substratum interactions . The experiment was performed by OPC cells being placed on PLA films and scaffolds modified with NH(2), PLL, or RGD in tissue culture media . OPC attachment to PLA films was assessed after 24 h of incubation . The growth and differentiation of the adherent OPCs on porous PLA scaffolds were assessed after 14 and 28 days for alkaline phosphatase (APase) activity and calcium levels, both of which increase as OPCs differentiate into mature bone cells . All assays were accomplished in triplicate, and data were tested with post hoc orthogonal contrasts (i.e., Fisher's least significant difference) at p < or = 0.05 . The PLA film surface-modified with RGD showed better OPC cell attachment than the other films . The cells on the PLA scaffolds surface-modified with RGD also exhibited an increase in APase activity and calcium levels in comparison with those on other scaffolds . This difference was apparent at both time intervals and was especially evident in the tissue culture media containing an osteogenic supplement . The results of this study indicate that modifying the surface of PLA polymer scaffolds with RGD enhances bone cell attachment and differentiation and may improve their ability to regenerate bone tissue more efficiently in wound models .

Circ Res, 2003 Feb 7, 92(2), 226 - 33
Regulation of cytokine-induced nitric oxide synthesis by asymmetric dimethylarginine: role of dimethylarginine dimethylaminohydrolase; Ueda S et al.; In response to vascular insults, inflammatory cytokines stimulate vascular smooth muscle cells (SMCs) to express an inducible isoform of nitric oxide synthase (iNOS) . Asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor, is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) . To determine whether the ADMA-DDAH system regulates cytokine-induced NO production, cultured rat SMCs were exposed to interleukin-1beta (IL-1beta) . IL-1beta (1 to 100 U/mL) dose-dependently stimulated not only iNOS but also DDAH expression and enzyme activity, accompanied by an increase in NO metabolite and by a decrease in ADMA content in culture media . A DDAH inhibitor (4124W, 5 mmol/L) augmented ADMA production (P<0.01) and decreased NO synthesis (P<0.01) in IL-1beta-stimulated SMCs . On the other hand, an adenovirus-mediated overexpression of DDAH reduced ADMA and enhanced NO production . Exogenous administration of NO donors (SNAP and SIN-1) dose-dependently increased NO metabolite in the culture media but had no effect on ADMA . Our results indicate two mechanisms of IL-1beta-induced NO synthesis: the direct stimulation of the expression of iNOS and the indirect stimulation of iNOS activity by upregulating DDAH and reducing ADMA . The ADMA-DDAH system may be another regulatory mechanism of inflammation-mediated NO production for human vascular diseases.

Am J Physiol Heart Circ Physiol, 2003 Jun, 284(6), H2227 - 34 Epub 2003 Feb 06.
Integrin shedding as a mechanism of cellular adaptation during cardiac growth; Goldsmith EC et al.; Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events . Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover . Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of beta(1)-integrin-immunoreactive material in the interstitium . Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of beta(1)-integrin . Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes . The beta(1)-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins . These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a beta(1)-integrin fragment from the cell surface . Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 169 - 74
Enhancement of monoclonal antibody production by lysine-containing peptides; Franek F et al.; In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine-containing peptides was compared . The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth-suppressing activity . No correlation of the production-enhancing activity with the character of the distribution of cell-cycle phases could be found . All of the tested peptides, including the negative control peptide Gly-Phe-Gly, altered the cell-cycle phases distribution in favor of the proportion of the S phase.The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids . Among the set of tested lysine-containing di- to pentapeptides, the best results were obtained with the tripeptide Gly-Lys-Gly . The growth-suppressing and production-enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect-a slight promotion of cell growth.

AIHA J (Fairfax, Va), 2003 Jan-Feb, 64(1), 40 - 7
A method for detecting fungal contaminants in wall cavities; Spurgeon JC; This article describes a practical method for detecting the presence of both fungal spores and culturable fungi in wall cavities . Culturable fungi were collected in 25 mm cassettes containing 0.8 microm mixed cellulose ester filters using aggressive sampling conditions . Both culturable fungi and fungal spores were collected in modified slotted-disk cassettes . The sample volume was 4 L . The filters were examined microscopically and dilution plated onto multiple culture media . Collecting airborne samples in filter cassettes was an effective method for assessing wall cavities for fungal contaminants, especially because this method allowed the sample to be analyzed by both microscopy and culture media . Assessment criteria were developed that allowed the sample results to be used to classify wall cavities as either uncontaminated or contaminated . As a criterion, wall cavities with concentrations of culturable fungi below the limit of detection (LOD) were classified as uncontaminated, whereas those cavities with detectable concentrations of culturable fungi were classified as contaminated . A total of 150 wall cavities was sampled as part of a field project . The concentrations of culturable fungi were below the LOD in 34% of the samples, whereas Aspergillus and/or Penicillium were the only fungal genera detected in 69% of the samples in which culturable fungi were detected . Spore counting resulted in the detection of Stachybotrys-like spores in 25% of the samples that were analyzed, whereas Stachybotrys chartarum colonies were only detected on 2% of malt extract agar plates and on 6% of corn meal agar plates.

J Orthop Res, 2003 Mar, 21(2), 320 - 5
Mitogens are increased in the systemic circulation during bone callus healing; Kaspar D et al.; The influence of mechanical tissue strain caused by flexible fracture fixation on the systemic occurrence of systemic mitogens during callus healing was investigated . For this purpose the mitogenic capacity and growth factor concentration of sera from patients undergoing fracture treatment were determined . Sera from 9 patients whose fractures had been stabilized by external fixation were collected before and during fracture treatment . The sera were added to cell culture media of the osteoblastic cell line SaOS-2 . After 5-6 days cell proliferation was measured . Transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-I (IGF-I) concentrations were analyzed in serum samples from different healing stages . STATISTICS: paired Wilcoxon-test . Sera from fracture patients decreased SaOS-2 proliferation in the first week after surgery (p<0.05) compared to sera obtained prior to surgery . In the fourth or fifth week proliferation increased significantly (p<0.03) . The increased proliferation of the SaOS-2 cells was associated with elevated levels of TGF-beta and IGF-I (p<0.05) . The higher mitogenic activity of sera suggests an increased level of circulating mitogens . In a previous study this increase had also been observed in patients during distraction osteogenesis treatment but not in patients with primary bone healing by a stable fixated plate . It is therefore assumed that their release from the fracture site is a consequence of mechanical stimulation by interfragmentary movement of fracture ends.

Theriogenology, 2003 Apr 15, 59(8), 1751 - 63
Effect of leukemia inhibitory factor on bovine embryos produced in vitro under chemically defined conditions; Sirisathien S et al.; The objective of these experiments was to assess putative embryotrophic effects of leukemia inhibitory factor (LIF) on bovine preimplantation development in chemically defined media . Recombinant human LIF was added to embryo culture media at a concentration of 100 ng/ml . When added for culture of morulae LIF had no positive effect on the proportion of embryos reaching the blastocyst stage . However, LIF significantly reduced development to the blastocyst stage when added for culture of 4-cell stage embryos (P<0.05) . In contrast, a positive effect was found for progression of blastocyst development . In vitro blastocyst hatching rates were significantly improved in the presence of LIF (P<0.02) . Number of total cells and of inner cell mass (ICM) cells were increased in LIF-treated blastocysts . In vitro survival of frozen-thawed blastocysts was not improved by adding LIF to morula stage embryos before cryopreservation . The pregnancy rate after direct transfer of cryopreserved LIF-treated embryos was not different from that for untreated control embryos . Data indicate that addition of LIF has no major beneficial effect on bovine embryos produced in these chemically defined conditions.

J Egypt Soc Parasitol, 1999, 29(1), 261 - 73
Study of factors affecting growth of Leishmania in cultures; Azab ME et al.; The rate of growth of Leishmania major and L . infantum in El-On's culture media supplemented with human, dog, rat and avian blood was studied in vitro . Rabbit blood was used as a control . The effect of culture with these types of blood on the infectivity of both Leishmania strains to albino mice was also studied . The results showed that a good yield of both L . major and L . infantum parasites can be obtained in culture by using avian blood as substitute for rabbit blood in El-ON's medium . In addition, rat blood gave good results with L . infantum . The morphological forms of L . major and L . infantum on all types of blood supplemented media: elongated promastigotes, spindle promastigotes, paramastigotes and amastigoes were present all through the culture period with variable percentages . The infectivity to experimental animals was not affected by culture of both Leishmania strains on rabbit, human, rat, dog as well as avian blood supplemented media.

Wei Sheng Yan Jiu, 2001 Jul, 30(4), 213 - 4
{Inhibitory effects of beta-carotene on hepatic cancer cell line SMMC-7721}; Luo W et al.; The inhibitory effect of beta-carotene on the proliferation of hepatic cancer was studied . Cells from a hepatic cancer cell line SMMC-7721 were incubated in culture media with 20, 40 and 80 mumol/L beta-carotene for 12, 24 and 48 h respectively . MTT test, Trypan blue exclusion test and DNA gel electrophoresis were used . The results of MTT test revealed that beta-carotene (20-80 mumol/L) could inhibit the proliferation of SMMC-7721 cells in a dose-dependent manner . DNA gel electrophoresis showed that the apoptosis of hepatic cancer cells could be induced by beta-carotene (40 mumol/L) . It is concluded that the proliferation of hepatic cancer cells inhibited by beta-carotene, probably through interfering DNA metabolism and inducing cell apoptosis.

J Orthop Sci, 2003, 8(1), 79 - 83
Thalidomide blocking of particle-induced TNFalpha release in vitro; Lin JH et al.; Tumor necrosis factor-alpha (TNFalpha) and interleukin-6 (IL-6), pleiotropic cytokines with osteotropic activities, are produced by multiple cells in the skeletal tissue, including macrophages and osteoblasts . They are thought to be pivotally involved in pathological bone resorption, such as that seen with aseptic loosening . Thalidomide is reported to have antiinflammatory, immunomodulatory effects in a number of inflammatory diseases . We investigated the effect of thalidomide on titanium (Ti) particle-induced TNFalpha and IL-6 production by both human macrophage U937 and osteoblast MG-63 cell lines . They were stimulated with 1 x 10(7) Ti particles/ml and treated simultaneously with or without various concentrations of thalidomide (from 2.5 ng/ml to 25 microg/ml) for 24, 48, or 72 h . Cell viability and proliferation were measured . TNFalpha and IL-6 in the supernatant of the culture media were also analyzed with an enzyme-linked immunosorbent assay . We found that with a concentration of thalidomide of less than 2.5 microg/ml the viability of the two cell lines did not differ significantly from that of controls treated simultaneously with 1 x 10(7) Ti particles/ml . Cell proliferation was inhibited to some extent when they were treated with thalidomide 2.5 microg/ml co-cultured with 1 x 10(7) Ti particles/ml . Thalidomide treatment was found to inhibit TNFalpha production in a dose-dependent manner in human macrophages exposed to Ti particles . At the clinically achievable drug dose of 2.5 microg/ml, 34.4% TNFalpha inhibition occurs . Thalidomide had no effect on IL-6 secretion in these cultures . These data support the idea that thalidomide may have potential for treating prosthetic loosening in humans.

Theriogenology, 2003 Apr 1, 59(7), 1641 - 9
Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos; Hyun SH et al.; In order to develop a culture system and recipient cytoplasm that could improve the developmental competence of somatic cell nuclear transfer (SCNT) embryos for successful cloning of pigs, we evaluated the effect of donor oocytes and in vitro maturation (IVM) media on maturation of oocytes and developmental competence of SCNT embryos . In Experiment 1, oocytes derived from sows or gilts were matured in two IVM media (TCM-199 versus NCSU-23) and maturation of oocytes was evaluated by the status of chromatin configuration, the diameter of matured oocytes, the thickness of the zona pellucida, and the size of the perivitelline space (PVS) . Sow oocytes matured in TCM-199 (S-TCM group) and NCSU-23 (S-NCSU group) showed significantly higher (P<0.05) maturation rates (S-TCM and S-NSCU, 86+/-4 and 82+/-4%, respectively) when evaluated by metaphase-II status than the gilt oocytes matured in TCM-199 (G-TCM group, 71+/-3%) and in NCSU-23 (G-NCSU-23 group, 71+/-3%) . Oocyte diameter, the thickness of the zona pellucida, and the perivitelline space of sow oocytes (S-TCM and S-NCSU) were larger than those of gilt oocytes (G-TCM and G-NCSU) after IVM (P<0.05) . In Experiment 2, SCNT was performed, using in vitro-matured oocytes from each group as recipient cytoplasm and porcine fetal fibroblasts as karyoplasts . The reconstructed embryos were electrically fused and activated, and cleavage and blastocyst formation were monitored under a stereomicroscope . The total cell number of flattened blastocysts stained with 5 microM bisbenzimide on day 7 were counted . In addition, in vitro matured non-enucleated oocytes were also electrically activated (parthenogenetic activation) and pronuclear formation was monitored . No difference in pronuclear formation rate after parthenogenetic activation and fusion rate after SCNT was observed among experimental groups . A significantly higher cleavage rate (P<0.05) was observed in S-TCM (69+/-4%) when compared with only G-NCSU (58+/-4%), but not with G-TCM (60+/-4%) or S-NCSU (68+/-4%) . The rate of blastocyst formation was significantly higher (P<0.05) in sow oocytes (24% in S-TCM and S-NCSU), when compared to that observed in G-TCM (15%), and G-NCSU (14%) . When the same source of oocytes was used, there was no significant difference in rate of blastocyst formation in the two culture media . Total cell number of blastocysts were not significantly different among experimental groups . In conclusion, the present study clearly demonstrated that sow oocytes have a greater developmental competence than gilt oocytes, regardless of the maturation medium examined.

Theriogenology, 2003 Apr 1, 59(7), 1567 - 74
Timing of nuclear maturation of nonstored and stored domestic cat oocytes; Katska-Ksiazkiewicz L et al.; In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O(2) atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes . One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10 degrees C for 16-24h before oocyte recovery . The second ovary from each pair was used as a nonstored control . In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O(2) atmosphere (a humidified gas atmosphere of either 5% CO(2) in air or 5% CO(2):5% O(2):90% N(2)) on IVM of both stored and nonstored oocytes . In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17-18, 20-21, 24-26, 28-30, 33-34 or 42-45 h before being evaluated for meiotic status . In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P>0.05) compared with nonstored . In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO(2) in air versus 5% CO(2):5% O(2):90% N(2)) affected oocyte maturation . In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17-18 h of culture was 36.1% and did not significantly increase (P>0.05) over time up to 28 h . The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42-45 h of culture . Therefore, we conclude that two "waves" of nuclear maturation of cat oocytes can be distinguished . The first wave takes place within 26 h and it is likely that most oocytes of this wave mature by 17-18 h; the second wave occurs after 28-30 h of IVM . It can be assumed that this double wave may reflect the presence of two oocyte populations with two different degrees of "prematuration" which require different lengths of IVM.

Invest Ophthalmol Vis Sci, 2003 Feb, 44(2), 521 - 8
Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells; Kaji Y et al.; PURPOSE: The corneal endothelium is a target of the aging process . This study was undertaken to reveal the relationship between corneal endothelial cell (CEC) death and the accumulation of advanced glycation end products (AGEs), by investigating the possible mechanism of accumulation of AGE in CECs and its effects on CEC death . METHODS: First, the in vivo expression of the receptor was investigated for AGE (RAGE) and galectin-3, both receptors for AGE, at both the mRNA and protein levels . Second, AGEs were added to the culture media of the cultured CECs, and the uptake of AGEs, the generation of reactive oxygen species, and the induction of apoptosis were investigated . RESULTS: Immunohistochemistry and RT-PCR demonstrated that both RAGE and galectin-3 were expressed in bovine CECs . After administration of AGE-modified bovine serum albumin to the culture medium, uptake of AGE was observed in the cytoplasm of the cultured bovine CECs . In addition, with increasing concentration of AGEs, the generation of reactive oxygen and the number of apoptotic cells also increased . CONCLUSIONS: These results show that the accumulation of AGEs in CECs induced apoptosis, in part, by increasing cellular oxidative stress . The accumulation of AGEs in the CECs of elderly patients may be involved in the loss of CECs during the aging process.

Anal Chem, 2003 Jan 15, 75(2), 255 - 60
Fluorophoric assay for the high-throughput determination of amidase activity; Henke E et al.; An assay has been developed for the high-throughput identification of amidase activity . Amines released from the enzyme-catalyzed hydrolysis of corresponding amides were detected by the formation of a fluorescent dye by coupling with 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole (NBD-CI) . Using this format, 22 lipases and esterases were tested for their ability to hydrolyze aromatic substituted N-acylamines in a microtiter plate format . Identified active enzymes were further characterized toward a broad range of compounds to determine the influence of substrate structure on activity . For recombinantly produced esterases, it could be shown that the assay works with high reproducibility and sensitivity, even in the presence of amino acids and proteins present in culture media and cell debris.

Neurochem Int, 2003 May, 42(6), 493 - 8
Increase in secretion of glial cell line-derived neurotrophic factor from glial cell lines by inhibitors of vacuolar ATPase; Nishiguchi M et al.; Glial cell line-derived neurotrophic factor (GDNF) was reported to be effective for treating subjects with neurodegenerative diseases such as Parkinson's disease . In search of finding a compound which promotes GDNF secretion, we found that concanamycin A (ConA), a vacuolar ATPase (V-type ATPase) inhibitor purified from Streptomyces diastatochromogens, enhanced GDNF secretion from glioma cells . The rat glioma cell line, C6, and the human glioma cell lines, U87MG and T98G, abundantly expressed GDNF mRNA, and secreted GDNF into culture media, and this event was potently enhanced by a Ca(2+) ionophore and by phorbol ester, as noted in other cells . ConA concentration dependently and potently increased GDNF release from C6, U87MG and T98G cells into culture media . In addition, ConA enhanced GDNF secretion from astrocyte primary cultures prepared from the human fetus with the same potency seen in glioma cell lines . Likewise, another V-type ATPase inhibitor, bafilomycinA1 facilitated GDNF release from C6, U87MG and T98G glioma cells, in a concentration-dependent manner . The potencies of these V-type ATPase inhibitors in enhancing GDNF secretion were consistent with those which inhibited V-type ATPase activity . These results suggest that blockade of V-type ATPase potently stimulates the secretion of GDNF from glial cells . The V-type ATPase inhibitors may be beneficial to use for the treatment of diseases in which increase in GDNF could be effective.

J Neurotrauma, 2002 Dec, 19(12), 1609 - 17
Intracerebral transplantation of marrow stromal cells cultured with neurotrophic factors promotes functional recovery in adult rats subjected to traumatic brain injury; Mahmood A et al.; This study was designed to examine the effects of bone marrow stromal cells (MSCs) cultured in vitro with or without neurotrophic factors transplanted into adult male Wistar rats after traumatic brain injury (TBI) . MSCs harvested from donor Wistar rats were cultured with either the culture medium containing brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) or the same culture media without these factors . Control and experimental animals were then traumatized by a controlled cortical impact . One day after the impact, either the placebo or the washed MSCs (1 x 10(6)) cultured with or without NGF and BDNF were transplanted adjacent to the site of injury . In addition, a nontreated group of rats was employed . Motor function of the animals was evaluated by the Rotarod test both before and after the injury . All animals were sacrificed 8 days after TBI, and the brain sections were stained by H&E as well as for immunohistochemistry . MSCs survived and migrated toward the injury site . The group treated with MSCs cultured with BDNF and NGF had a significantly higher number of engrafted cells than the group treated with MSCs cultured without BDNF and NGF (6.3 x 10(4) +/- 4250 compared to 4.1 x 10(4) +/- 3684; p < 0.05) . In both groups, some transplanted MSCs showed positive staining for astrocytic (GFAP) and neuronal markers (Neu N and MAP-2) . The groups treated with MSCs had better motor function than the groups receiving no treatment or receiving the placebo (PBS; p < 0.05); however, the improvement reached statistical significance only in the group treated with MSCs cultured with neurotrophic factors . These data suggest that more robust motor function described in rats subjected to TBI and treated with intracerebral transplantation of MSCs was achieved by the use of MSCs cultured with neurotrophic factors.

Eur J Neurosci, 2003 Jan, 17(2), 205 - 11
Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF-1 as a survival factor; Perez-Martin M et al.; Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors . In this study we have characterized the nature of new cells and have assessed whether the insulin-like growth factor-1 (IGF-1) receptor regulates their survival and/or proliferation . The explants were composed of the ependymal layer and attached subependymal cells . Ependymal cells in culture were labelled with glial markers (S-100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media . Most subependymal cells were immunoreactive for beta III-tubulin, a neuronal marker, and did incorporate bromodeoxiuridine . Subependymal neurons displayed immunoreactivity for IGF-1 and its receptor and expressed IGF-1 mRNA, indicating that IGF-1 is produced in the explants and may act on new neurons . Addition to the culture media of an IGF-1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells . However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF-1 receptor is involved in the survival of subependymal neurons . In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF-1 is locally produced in the explants and may regulate the survival of the proliferating neurons.

Toxicol In Vitro, 2003 Feb, 17(1), 41 - 7
Phagocytosis of titanium particles and necrosis in TNF-alpha-resistant mouse sarcoma L929 cells; Osano E et al.; In the oral cavity, titanium is an excellent biocompatible material . However, it is reported that high ratios of intracellular titanium particles can cause cell apoptosis or necrosis by as-yet unknown mechanisms . The purpose of this study was to investigate the response of tumor necrosis factor alpha (TNF-alpha)-resistant L929 fibroblasts to titanium particles . Cells were cultured in Eagle's medium supplemented with fetal bovine serum and L-glutamine . Titanium particle sizes were less than 9 micro . Cytotoxicity was assayed by a cell counting kit, trypan blue dye exclusion test and lactate dehydrogenase (LDH) leakage . The production of reactive oxygen species (ROS) was detected by a confocal laser scanning microscope (CLSM) using dichlorofluorescein diacetate as a fluorescent probe . Morphology was viewed by a CLSM and with an X-ray microanalyser (XMA) . When titanium particles were added to cells, the viability decreased to around 50% at a particle concentration of 2.0% . The number of dead cells and LDH activity in the culture media increased significantly between 1 and 2 days . However, formation of active oxygen species did not occur, since no dichlorofluorescein fluorescence was observed . A scanning electron photomicrograph (SEM) revealed a large number of particles covering or adhering to cellular components in lysed cells compared with flattened control cells attached to the substrate . The XMA showed that the titanium accumulation was coincident with the deformed cell shape . The CLSM also confirmed that particles were within the cells . From these results it was concluded that titanium particles ingested in large quantities into the cell induced necrosis by a pathway other than by producing ROS.

Reprod Biomed Online, 2001, 2(2), 113 - 119
Growth of human preimplantation embryos in vitro; Mehta RH; The human oocyte fertilizes and develops into embryos in the Fallopian tube and reaches the uterus only after compaction . However, for several years embryos that were developed following in-vitro fertilization (IVF) were transferred into the uterus on day 2 or 3 at the 4-8 cell stage in contrast to the in-vivo situation where they would be present in the Fallopian tube . Earlier attempts to grow embryos in vitro for 5 to 6 days were not always successful . Attempts were therefore made to understand the in-vivo environment of the Fallopian tube where the early embryonic development occurs . This article reviews the studies carried out to understand the composition of fluids in the Fallopian tube specifically with reference to the energy metabolites - lactate, pyruvate and glucose; it also covers how the formulation of culture media for human IVF and embryonic development were modified over the years based on some classical work done on embryo culture in laboratory animals.

Fa Yi Xue Za Zhi, 2000 Aug, 16(3), 129 - 31, 190
{The effect of injuries on Fn synthesized by FBs and its implication in wound timing}; Shen Y et al.; cFn synthesized by FBs around the wound was detected by immunochemistry in vitro . Using the method of ELISA, The concentration of cFn in the culture media was observed . The concentration of cFn had no change within 30 minutes after injury, but started to increase after 1 hour of the injury and kept this trend within next five hours after injury . By static analysis, it can be obtained that the concentration of cFn in culture media is time-dependent after injury.

Atherosclerosis, 2003 Feb, 166(2), 291 - 301
Induction of cellular glutathione and glutathione S-transferase by 3H-1,2-dithiole-3-thione in rat aortic smooth muscle A10 cells: protection against acrolein-induced toxicity; Cao Z et al.; There is increasing evidence that aldehydes, including acrolein generated endogenously during the degradation process of biological molecules or the metabolism of foreign chemicals may be involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis . Because glutathione (GSH) and GSH S-transferase (GST) are a major cellular defense against the toxic effects of reactive aldehydes, in this study we have characterized the inducibility of GSH and GST by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) and their protective effects against acrolein-induced toxicity in rat aortic smooth muscle A10 cells . Incubation of rat aortic A10 cells with micromolar concentrations of D3T resulted in a concentration- and time-dependent induction of both GSH and GST . Treatment of A10 cells with D3T also led to induction of gamma-glutamylcysteine synthetase, the key enzyme involved in GSH biosynthesis . Notably, the levels of GSH and GST remained higher than basal levels 72 h after removal of D3T from the culture media . To examine the protective effects of D3T-induced GSH and GST against reactive aldehyde-mediated toxicity, A10 cells were pretreated with D3T and then exposed to acrolein . Pretreatment of A10 cells with D3T resulted in a marked decrease of acrolein-induced toxicity as determined by 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide reduction assay and morphological changes . To further demonstrate the involvement of GSH and GST in protecting against acrolein-induced toxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively . Either depletion of cellular GSH by BSO or inhibition of cellular GST by sulfasalazine led to a marked potentiation of acrolein-induced toxicity in A10 cells . Furthermore, co-treatment of cells with BSO was found to greatly abolish the protective effects of D3T on acrolein-induced toxicity . Taken together, our results demonstrate for the first time that both GSH and GST in aortic smooth muscle cells can be induced by D3T, and that this increased cellular defense affords great protection against reactive aldehyde-induced cardiovascular cell injury.

Theriogenology, 2003 Mar, 59(5-6), 1393 - 402
Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts; Mtango NR et al.; The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts . In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation (n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1) . medium only control; (2) . activin (10 ng/ml); (3) . epidermal growth factor (EGF) (10 ng/ml); (4) . insulin 5 microg/ml and (5) . GH (100 ng/ml) . There was an increase (P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls . There was no significant difference between insulin and control groups . A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone . After thawing, embryos were cultured in five treatments groups as described above . Groups EGF and GH had higher (P<0.05) survival rates with a mean blastocyst survival of 95.0+/-1.5 and 93.1+/-3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3+/-3.4 and 62.0+/-3.2%, respectively) . Thawed control blastocysts had a mean cell count of 52.7+/-3.3% . With the exception of insulin, all growth factors and GH tested showed higher (P<0.01) total cell numbers when compared to controls . In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.

Int J Dev Neurosci, 2002 Dec, 20(8), 607 - 17
Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine; Cubillos S et al.; The use of culture media of known composition are necessary for studying the role of trophic molecules . Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum . After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined . Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used . The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites . Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants . However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum . Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine . The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum.

Int J Dev Neurosci, 2002 Dec, 20(8), 593 - 606
D1 dopamine receptor regulation of cell cycle in FGF- and EGF-supported primary cultures of embryonic cerebral cortical precursor cells; Zhang L et al.; In the mammalian fetus, proliferation of the majority of cells destined for the cerebral cortex takes place within the transient proliferative zones of the cerebral wall . Recent investigations have demonstrated that cell of these zones express high levels of D1 dopamine receptors (D1Rs) . However, the specific roles of these receptors have not been investigated . The present study tests the hypothesis that D1Rs are capable of regulating the cell cycle of cerebral cortical precursor cells . For this purpose, primary cultures of cells of the proliferative zones from the cerebral wall of 14-day-old mouse fetuses were generated and maintained in the presence of either fibroblast growth factor-2 (FGF2) or epidermal growth factor (EGF) . These growth factors were chosen as supporting two distinct populations of precursor cells in the fetal cortical proliferative matrix . The involvement of D1Rs in the regulation of proliferative activity was examined by the addition of a range of concentrations of the D1R-specific agonist, SKF82958, to the culture media . Bromodeoxyuridine incorporation assays demonstrated that exposure to this agonist led to a dose-dependent reduction of DNA synthesis in both FGF2- and EGF-supported cultures . Flow cytometric cell cycle assays further revealed that this was due to prevention of the transition of cells from the G1 phase to the S phase of the cell cycle . The D1R specificity of the effects of SKF82958 was supported in that they were blocked by the addition of the D1R antagonists, SCH23390 or NNC010756 . We also found that D1R stimulation induced stronger suppression of proliferative activity in EGF-supported than in FGF2-supported cultures . Our observations suggest that D1Rs are capable of regulating the cell cycle during corticogenesis . Furthermore, they raise a possibility that these receptors may display different efficacies in affecting proliferative activity in FGF2-supported versus EGF-supported cerebral cortical precursor cells.

Wei Sheng Yan Jiu, 2001 May, 30(3), 139 - 41
{Developmental toxicity of Al(NO3)3 on rat embryos}; Zhang B et al.; The developmental toxicity and its mechanism of Al(NO3)3 on embryos in SD rats were studied . On the 9.5th day of gestation, the embryos were incubated in a whole-embryo culture system with Al(NO3)3 at concentrations of Al3+ from 0.6 to 9.0 micrograms/ml in culture media for 48 hrs . Viable embryos were evaluated by Brown's morphological scoring system, and the diameter of yolk sac, crown-rump, head length and the dry weight of embryos were measured . There was a dose-dependent relations of decreasing embryo development with increasing concentrations of Al3+ . Yolk sac diameter, head length, dry weight and heart, forelimb as well as neural tube scores decreased significantly at 1.2 micrograms/ml (P < 0.05) . When embryos were exposed to Al3+ at > or = 3.0 micrograms/ml, the embryonic development and morphological differentiation were obviously inhibited(P < 0.05); meanwhile, the incidence of dysmorphogenesis significantly increased, including neural tube defects and dorsiflexion teratogenesis . The results suggested that aluminum might be a developmental toxicant and dysmorphogenesis agent.

Arterioscler Thromb Vasc Biol, 2003 Jan 1, 23(1), 52 - 7
Peroxisome proliferator-activated receptor gamma ligands increase release of nitric oxide from endothelial cells; Calnek DS et al.; OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduce lesion formation in animal models of atherosclerosis by mechanisms that have not been defined completely . We hypothesized that PPARgamma ligands stimulate endothelial-derived nitric oxide release (*NO) to protect the vascular wall . METHODS AND RESULTS: The PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) or ciglitazone, stimulated a PPAR response element-luciferase reporter construct in transfected porcine pulmonary artery endothelial cells (PAECs), demonstrating that PPARgamma was transcriptionally functional . Treatment with 15d-PGJ2 or ciglitazone significantly increased release of *NO from PAECs or human aortic endothelial cells and augmented calcium ionophore-induced *NO release from human umbilical vein endothelial cells measured by chemiluminescence analysis of culture media . Increases in *NO release caused by treatment with 15d-PGJ2 occurred at 24 hours, but not after 1 to 16 hours, and were abrogated by treatment with the transcriptional inhibitor alpha-amanitin . Overexpression of PPARgamma or treatment with 9-cis retinoic acid also enhanced PAEC *NO release . Neither 15d-PGJ2 nor ciglitazone altered eNOS mRNA, whereas 15d-PGJ2, but not ciglitazone, decreased eNOS protein . CONCLUSIONS: Taken together, these findings demonstrate that PPARgamma ligands stimulate *NO release from endothelial cells derived from multiple vascular sites, through a transcriptional mechanism unrelated to eNOS expression.

Fertil Steril, 2003 Jan, 79(1), 146 - 50
Secretion of granulocyte chemotactic protein-2 by cultured human endometrial stromal cells; Mine S et al.; OBJECTIVE: To evaluate the expression of granulocyte chemotactic protein-2 (GCP-2) in human endometrial stromal cells . DESIGN: The effects of interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma, and lipopolysaccharide (LPS) on the production of GCP-2 by endometrial stromal cells were investigated . SETTING: Research laboratory at a medical university . PATIENT(S): Eight endometrial specimens in the late proliferative phase were used . INTERVENTION(S): Endometrial stromal cells were incubated for 24 hours with IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, IFN-gamma, and LPS.The concentration of GCP-2 in the culture media was measured using an enzyme-linked immunosorbent assay (ELISA) . RESULT(S): A small amount of GCP-2 was detected in the culture media of unstimulated endometrial stromal cells . The production of GCP-2 by endometrial stromal cells was stimulated with IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, and LPS in a dose-dependent manner . Interferon-gamma did not affect GCP-2 production by these cells . CONCLUSION(S): These results suggest that GCP-2 is an additional ELR(+)-CXC chemokine expressed in endometrial stromal cells . The modulation of GCP-2 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating the neutrophil trafficking in the endometrium.

Fertil Steril, 2003 Jan, 79(1), 81 - 90
Strategies and outcomes of the first 100 cycles of preimplantation genetic diagnosis at the Guy's and St . Thomas' Center; Pickering S et al.; OBJECTIVE: To establish strategies for the implementation of a successful preimplantation genetic diagnosis (PGD) service . DESIGN: Retrospective review of data from a single center . SETTING: A United Kingdom National Health Service hospital . PATIENT(S): Patients (60 couples) were referred for PGD from UK genetic centers . INTERVENTION(S): We followed the protocol of ovarian stimulation, oocyte retrieval, fertilization, single cell biopsy on day 3, and embryo transfer on day 4 . Pregnancies unaffected by the familial genetic condition . RESULT(S): A total of 60 couples was treated for 20 different conditions . Early cycles using nonsequential embryo culture media were less successful (13% pregnancy rate/embryo transfer) than later cycles using sequential media (33.5%) . Ninety-four percent of embryos (n = 473) had a single cell removed at biopsy . The overall pregnancy rate was 24% per cycle started, 29% per egg collection, 38% per transfer, and 40% per couple treated . In one cycle, an affected pregnancy followed PGD for spinal muscular atrophy (SMA) . CONCLUSION(S): The use of sequential media and single cell biopsy results in a successful PGD program with encouraging pregnancy rates.

J Clin Endocrinol Metab, 2003 Jan, 88(1), 484 - 92
Stromal cells of the human postmenopausal ovary display a distinctive biochemical and molecular phenotype; Jabara S et al.; The stroma of the human postmenopausal ovary is postulated to produce androgens, but evidence for and against this idea exits in the literature . The purpose of this study was to determine whether key steroidogenic enzymes involved in androgen synthesis are expressed in the postmenopausal ovarian stroma . Stromal cells were isolated from postmenopausal ovaries and expression for genes involved in steroidogenesis {steroidogenic acute regulatory protein (StAR), P450scc, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) P450c17, and P450c27} as well as for several growth factor binding proteins {gremlin, IGF binding protein-4, follistatin, and secreted frizzled-related protein (sFRP)-1 and -4}, were compared with cultured human theca cells and dermal fibroblasts . Production of steroids (pregnenolone, progesterone, and hydroxysterol metabolites) and the metabolism of {3H} pregnenolone by ovarian stromal cells were also assessed . Isolated ovarian stromal cells from different subjects had a uniform morphology within and across cultures . Quantitative real time RT-PCR revealed that StAR, P450scc, and 3 beta-HSD transcripts were, respectively 30, 25, and 45 times more abundant in theca cells than in stromal cells . Mean levels of P450scc and 3 beta-HSD transcripts in stromal cells were similar to those found in dermal fibroblasts, whereas StAR transcripts in stromal cells were 285-fold more abundant than in fibroblasts . There was no significant expression of P450c17 in ovarian stromal cells or fibroblasts ( approximately 2000-fold less than in theca cells) . Western analysis demonstrated the presence of the 30-kDa StAR mature protein in the cultured stromal cells, whereas P450c17 protein was not detectable . Ovarian stromal cells did not metabolize {3H} pregnenolone in the presence or absence of 8-Br-cAMP . Furthermore, pregnenolone and progesterone secretion by stromal cells was also undetectable, even in the presence of 22-hydroxycholesterol . P450c27 protein was detected in ovarian stromal cells and its metabolic products (i.e . 27-hydroxycholesterol and cholestenoic acid) were found in the culture media, reflecting functional cholesterol 27-hydroxylase activity . Follistatin, gremlin, IGF binding protein-4, and sFRP-1 and -4 transcripts were detected in the stromal cells in relative amounts significantly higher than theca cells, but not significantly different from fibroblasts, except for sFRP-1, which was significantly higher in stromal cells . Our observations demonstrate that stromal cells of the postmenopausal ovary have a signature biochemical and molecular phenotype that can be distinguished from fibroblasts . These cells do not appear to have significant steroidogenic potential in vitro, but they do metabolize cholesterol into hydroxysterols . We conclude that the predominant stromal cells of the postmenopausal ovary are not a significant site of androgen biosynthesis.

Cancer Res, 2003 Jan 1, 63(1), 214 - 21
Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma; Song P et al.; It is well established that small cell lung carcinomas (SCLCs) express receptors for acetylcholine (ACh) and that stimulation of these receptors by nicotine or other cholinergic agonists stimulates cell growth via activation of nicotinic cholinergic receptors (nAChRs) and/or muscarinic cholinergic receptors (mAChRs) . The aim of this study was to determine whether SCLC cells synthesize and secrete ACh and respond to endogenous ACh to create a functioning cholinergic autocrine loop . Reverse transcription-PCR was used to screen a panel of SCLC cell lines for components of cholinergic signaling . Choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT), as well as alpha3, alpha5, alpha7, beta2, and beta4, nAChR subunits and M3 and M5 mAChRs, were found to be present in most of the SCLC cell lines tested . Real-time PCR showed that mRNA levels for ChAT, VAChT, and alpha7 and beta2 nAChR subunits varied significantly among different SCLC cell lines tested . The H82 cell line was found to express the highest levels of ChAT, and that cell line was chosen for additional studies of ACh release and cell proliferation . ACh was easily detectable in H82 cell culture media, and levels of ACh were increased by the acetylcholinesterase inhibitor neostigmine . Vesamicol, an inhibitor of VAChT, and hemicholinium-3, an inhibitor of choline transport, both reduced H82 cell ACh basal release in a dose-dependent manner . In parallel with the reductions of ACh release, vesamicol and hemicholinium-3 also decreased H82 cell proliferation . H82 cell proliferation was also inhibited by the muscarinic and nicotinic antagonists atropine and mecamylamine, respectively, in dose- and time-dependent manners . Finally, archival cases of SCLC were screened by immunohistochemistry for expression of ChAT . Thirteen of 26 tumors screened were positive for ChAT . These findings demonstrate that SCLC can synthesize, secrete, and degrade ACh and that released ACh stimulates SCLC cell growth . Identification of this new autocrine loop provides a potential new target for therapeutic intervention.

Theriogenology, 2003 Feb, 59(3-4), 939 - 49
Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development; Ali AA et al.; Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers . In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period . Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD) . Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation . However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development . During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05) . In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development . Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.

Theriogenology, 2003 Feb, 59(3-4), 719 - 34
Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig; Nguyen VT et al.; The objective of this study was to investigate the effects of osmolarity of culture media on the development of porcine parthenogenetic diploids . Oocyte-cumulus-granulosa cell complexes were collected from ovaries and then in vitro-cultured for 48 h . The mature oocytes were subjected to a single electro-stimulation (El-St; 100 micros, 1500 V/cm), treated with 5.0 microg/ml Cytochalasin B for 4h and then cultured under various conditions as described below . In Experiment 1, the diploids were cultured for 168 h after El-St in modified Whitten's medium with 256 mOsmol (mWM256), mKRB with 309 mOsmol, and mWM with 309 mOsmol (mWM309), in which the osmolarity was adjusted by addition of NaCl or mannitol, or by reduction of distilled water . In Experiment 2, the diploids were cultured in the five media used in Experiment 1 for the first 48 h, and then in mWM256 until 168 h after El-St . In Experiment 3, the diploids were cultured for the first 48 h in mWM with osmolarity adjusted from 256 to 330 mOsmol by addition of NaCl for the first 48 h and then in mWM256 until 168 h after El-St . In Experiment 4, the diploids were cultured in mWM with 290 mOsmol (mWM290) for the first period of 24, 48, or 72 h, and then in mWM256 until 168 h after El-St . In Experiment 5, after diploids were cultured in mWM290 for the first 48 h, the obtained 4-cell diploids were transferred to mWM with osmolarity adjusted from 200 to 310 mOsmol by addition of NaCl, then cultured until 168 h after El-St . All media were supplemented with 0.5mg/ml hyaluronic acid and 4.0mg/ml bovine serum albumin . The results obtained in Experiments 1-5 indicate that the osmolarity of a medium, but not the Na(+)/K(+) ratio, exerts effects on the development of diploids to the blastocyst stage . The change of osmolarity of the culture media after the 4-cell stage increased the rate of expanded blastocyst formation in porcine diploids . The optimal osmolarities of culture medium for the first 48 h after El-St (before the 4-cell stage) were 290 and 280-320 mOsmol, and those for the later period (after the 4-cell stage) were 256 and 220-270 mOsmol, respectively.

Zhonghua Shao Shang Za Zhi, 2002 Feb, 18(1), 32 - 3
{An experimental study of the inhibitory effects of Tripterygium extract (LLZ) on hypertrophic scar - derived fibroblasts}; Xie W et al.; OBJECTIVE: To explore the effects of Tripterygium extract (LLZ) on postburn hypertrophic scar - derived fibroblasts, so as to provide us an experimental support for the treatment of postburn hypertrophic scar . METHODS: Hypertrophic scar - derived fibroblasts were harvested and cultured in vitro . A serial concentrations of LLZ (5 x 10(-3), 5 x 10(-4), 5 x 10(-5), 5 x 10(-6) g/L) were added to the culture media . The cellular morphology, proliferating activity and the cytotoxicity of the drug were observed after 24 hours of culture . RESULTS: The fibroblast morphology could be altered by any concentration of LLZ solution . Furthermore, the cell count and the cellular proliferating activity were all decreased obviously by the treatment of LLZ . CONCLUSION: The cell morphology and proliferation activity of postburn scar-derived fibroblasts could all be inhibited by LLZ, and it was the result of its toxic effects.

Ann Acad Med Stetin, 2001, 47, 107 - 24
{Improved method for delivery room collection and storage of human cord blood cells for grafting}; Szolomicka-Kurzawa P; Haematopoietic stem cell transplantation is indicated in several haematologic and genetic diseases, the most notable being aplastic anemia and leukemias . Bone marrow has been the traditional source of these cells . Human umbilical cord blood (UCB) has recently become an alternative source of haematopoietic stem cells for transplants . The advantages of cord blood include noninvasive collection without risk to mother and neonate, low risk of viral infection, and immunologic immaturity of cord cells . Single umbilical cord blood donation is usually sufficient for transplantation to adult recipients . Additionally, banking of HLA-typed UCB appears valuable in patients lacking a family donor . This study has focused on basic "perinatological" parameters of umbilical cord blood: average volume of single donation UCB and initial storage conditions before isolation of haematopoietic stem cells . Additionally, the mean content of CD34+ haematopoietic stem cells in leukocyte, lymphocyte and mononuclear cell fractions was established . Correlations between levels of so-called pro-inflammatory cytokines (present in cord blood serum) and number, viability and clonogenicity of cord blood mononuclear cells were checked . UCB samples were obtained by "open" collection during vaginal deliveries and cesarean sections . The collected blood was stored in solutions of anticoagulants (ACD, CPDA-1, heparin) and culture media (PBS, Iscove medium, RPMI), during several time intervals (0-1 h, 1-6 h, 6-12 h, 12-24 h) and at two temperatures (+4 degrees C, ambient) . UCB volumes, as well as MNC counts, correlated with delivery type, placental weight, neonatal body weight and duration of pregnancy . The concentration, viability and clonogenicity of MNCs were assessed after collection and storage . The subpopulation of CD34+ haematopoietic stem cells was isolated from MNCs using monoclonal antibodies and magnetic-based separation . The number, viability and clonogenicity of CD34+ cells were evaluated . Subsequently in some samples, the concentration of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha), number of mononuclear cells and in vitro clonogenicity of myeloid progenitors (CFU-GM) were determined . It was found that the collected blood volume depended on neonatal body weight (Fig . 1) . Umbilical blood could be stored either at ambient temperature (Fig . 4) or +4 degrees C (recommended because of reduced risk of infection) for up to 24 hours in RPMI solution (Fig . 5) with heparin (Fig . 2, 3) . CD34+ cell count correlated with mononuclear cell count only (Fig . 6) . A negative correlation between the number of mononuclear cells and concentration of TNF-alpha was revealed (Fig . 7), as well as between the number of detectable CFU-GM and concentration of IL-1 beta (Fig . 8) . In conclusion, UCB collection and short-term storage is a safe and simple method for graftable haematopoietic stem cell recovery . Save for IL-1 beta and TNF-alpha, cytokine levels did not correlate with the studied parameters of umbilical cord blood.

J Androl, 2003 Jan-Feb, 24(1), 120 - 30
Establishment and characterization of neonatal mouse sertoli cell lines; Hofmann MC et al.; Sertoli cells isolated from 6-day postpartum mouse testes were conditionally immortalized with the simian virus 40 large tumor antigen gene (SV40-LTAg) under the control of a promoter inducible with ponasterone A, an analog of ecdysone . This strategy produced 2 cell lines, which exhibited mixed phenotypes . We first tested the conditional expression of the LTAg gene in the presence or absence of ponasterone A . The results showed that both cell lines expressed LTAg when the inducer was present in the culture media . When ponasterone A was removed, the majority of the cells died . After 60 generations, however, the continued expression of LTAg in the absence of the hormone indicated that unknown changes may have occurred in the genome of the cells . One of the cell lines was further subcloned, resulting in 7 new lines exhibiting a morphology resembling that of Sertoli cells in tissue culture . Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on RNA collected from each cell line in order to determine which cells were phenotypically similar to Sertoli cells in vivo . All cell lines expressed the products of the Sertoli cell-specific genes stem cell factor (SCF) and sulfated glycoprotein-2 (SGP-2), in addition to alpha-inhibin, GATA-1, and steroidogenic factor-1 . Further, the lines express growth and differentiation factors known to act upon germ cells in vivo and in vitro such as leukemia inhibitory factor (LIF), transforming growth factor beta (TGF-beta), and basic fibroblast growth factor (bFGF) . Moreover, when used as feeder layers in cocultures, at least 2 of these lines are able to maintain the viability of type A spermatogonia for at least 7 days and to support the first steps of spermatogonial differentiation.

Appl Environ Microbiol, 2003 Jan, 69(1), 56 - 65
ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression; Aro N et al.; We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1 . Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression . Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed . On cellulose-based medium, on which cellulases are needed for growth, the Deltaace1 strain grew better than the host strain due to the increased cellulase production . On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes . A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Deltaace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2002 Feb, 10(1), 47 - 51
{The effects of sera from patients with paroxysmal nocturnal hematoglobinuria on the growth of CD34(+) cells}; Han B et al.; To investigate the effects of sera on the growth of single CD34(+) cells from patients with paroxysmal nocturnal hematoglobinuria (PNH), sera from both PNH patients and normal individuals were added separately to the single cell culture system and semi-solid colony formation system . The growth of the normal CD34(+) and PNH CD34(+) CD59(+) and CD34(+) CD59(-) cells was evaluated . No growth difference was found for growth of the normal CD34(+) and PNH CD34(+) CD59(+) cells when PNH or normal sera were added to the culture media in either single cell culture or colony formation culture . While no difference was detected for single PNH CD34(+) CD59(-) cells growth when PNH or normal sera were added, more colonies were observed in semi-solid culture with PNH sera . A conclusion was reached that compared with those from normal controls, the sera from PNH patients had no significant influence on single hematopoietic stem/progenitor cells derived from normal subjects and from PNH patients, but the PNH sera might promote the colony formation of the CD34(+) CD59(+) cells in semi-solid culture

J Dairy Sci, 2002 Dec, 85(12), 3277 - 86
Evidence for a local effect of leptin in bovine mammary gland; Silva LF et al.; On average, high-energy diets promoting body growth rates above 1 kg/d before puberty impair mammary development by 15 to 20% in cattle . We hypothesized that leptin, a protein produced by adipocytes, mediates the inhibitory effect of high-energy diets on mammary development . Therefore, our objectives were to determine the effect of leptin on mammary epithelial cell proliferation, and the distribution of mRNA for two leptin receptor isoforms in prepubertal bovine mammary glands and other peripheral tissues . Addition of leptin to culture media containing either 5 ng/ml of insulin-like growth factor-I (IGF-I) or 1% fetal bovine serum decreased DNA synthesis of a bovine mammary epithelial cell line (MAC-T) in a dose-dependent manner . The minimal doses of leptin that decreased IGF-I- and fetal bovine serum-stimulated cell proliferation were 64 and 1 ng/ml, respectively . In addition, we determined that MAC-T cells and isolated bovine mammary epithelial cells express the long form of leptin receptor (Ob-Rb) mRNA . Ob-Rb mRNA was detected in all bovine tissues examined . In contrast with reports on other species, mRNA expression of the short form of leptin receptor (Ob-Ra) was detected only in bovine liver, pituitary body, and spleen . These results support the concept that leptin mediates the inhibitory effect of high-energy diets on mammary development.

Acta Microbiol Immunol Hung, 2002, 49(4), 445 - 54
Bioconversion of poultry wastes . I--Factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi; Abdel-Fattah GM et al.; The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus, A . flavus and Trichoderma sp . was at 30 degrees C . The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp . The optimum pH was at 6.4 for A . terreus and pH 6.6 for A . flavus and Trichoderma sp . The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste . The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A . terreus, A . flavus and Trichoderma sp., respectively . Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi . The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, although no significantly increased uricase production was found.

Cardiovasc Res, 2003 Jan, 57(1), 232 - 7
Adrenomedullin augments the release and production of tissue factor pathway inhibitor in human aortic endothelial cells; Marutsuka K et al.; OBJECTIVES: Adrenomedullin (AM) is a hypotensive and vasodilative peptide . It has been reported that AM plays several biological roles in cardiovascular and endocrine systems, however, effects on the blood coagulation system have not been examined . In this study, we examined its effect on tissue factor pathway inhibitor (TFPI), which is a potent inhibitor of tissue factor/factor VIIa complex-induced coagulation cascade, and is synthesized and constitutively secreted by endothelial cells (ECs) . The aim of this study was to elucidate the effects of AM on release and production of TFPI in ECs . METHODS: Cultured human aortic ECs were incubated with AM (10(-14)-10(-6) M), and the antigen levels and activity of the free and total form of TFPI after AM exposure were measured in culture media using ELISA . Furthermore, receptor interaction and cellular signaling of AM were investigated . RESULTS: AM augmented TFPI release and production in a dose-dependent manner . The effect on TFPI production was inhibited by AM receptor antagonist (AM22-52), the monoclonal antibody against C-terminal region of AM, MAPKK inhibitor, and cAMP antagonist . CONCLUSION: These findings indicate that AM might play an important role in the modulation of anticoagulant properties in blood circulation.

J Biol Chem, 2003 Mar 14, 278(11), 9698 - 705 Epub 2002 Dec 24.
Lipoprotein lipase affects the survival and differentiation of neural cells exposed to very low density lipoprotein; Paradis E et al.; Lipoprotein lipase (LPL) is a key enzyme involved in the metabolism of lipoproteins, providing tissues like adipose tissue or skeletal muscle with fatty acids . LPL is also expressed in the brain, fulfilling yet unknown functions . Using a neuroblastoma cell line transfected with a NEO- or a LPL-expression vector, we have developed a model to study the function of LPL in neurons exposed to native or copper-oxidized lipoproteins . The addition to the culture media of VLDL with 10 microm copper sulfate led to a significant reduction in the viability of NEO transfectants whereas LPL-transfectants were protected from this injury . In the presence of VLDL and CuSO(4), LPL transfectants were even able to display significant neurite extension . This neuritogenic effect was also observed in LPL transfectants exposed to native lipoproteins . However, addition of VLDL particles oxidized with CuSO(4) prior to their addition to the culture media resulted in neurotoxic effects on LPL transfectants . These findings suggest that the presence of LPL in cultured neuronal cells modulates the physiological response of neurons following exposure to native or oxidized lipoproteins . LPL could thus play a key role in the differentiation of Neuro-2A cells and in the pathophysiological effects of oxidative stress in several neurodegenerative disorders.

Am J Obstet Gynecol, 2002 Dec, 187(6), 1574 - 80
Modulation of trophoblast function by tumor necrosis factor-alpha: a role in pregnancy establishment and maintenance?
Monzon-Bordonaba F, Vadillo-Ortega F, Feinberg RF.
OBJECTIVE: Trophoblast differentiation is a critical process for successful implantation and establishment of the human placenta . The aim of this study was to characterize the effect of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) on the expression of markers of trophoblast function and differentiation.STUDY DESIGN: Human cytotrophoblasts were stimulated with 1 and 10 ng/mL recombinant TNF-alpha or IL-6 . Cell viability was determined and conditioned culture media was analyzed by gelatin zymography to assess protease secretion and by enzyme-linked immunosorbent assays to measure production of beta-human chorionic gonadotropin and oncofetal fibronectin . RESULTS: TNF-alpha increased secretion of urokinase-type plasminogen activator up to 3-fold of basal unstimulated production . Stimulation of cytotrophoblasts with this cytokine also inhibited beta-human chorionic gonadotropin secretion up to 75% . TNF-alpha did not modify the secretion of matrix metalloproteinase-9 and oncofetal fibronectin . IL-6 had no effect on these trophoblast differentiation markers . CONCLUSION: These results show that TNF-alpha stimulated cytotrophoblasts modulate the expression of differentiation markers, down-regulating the autocrine signals that promote syncytialization, and increasing their invasive capacity through up-regulation of proteases . We suggest that this regulatory mechanism of trophoblast function could play an important role during trophoblast implantation, in pregnancy failure and in the normal and pathologic rupture of fetal membranes.

Tumour Biol, 2002 Jul-Aug, 23(4), 202 - 11
The enzymatic basis for the conversion of nonfucosylated to fucosylated alpha-fetoprotein by acyclic retinoid treatment in human hepatoma cells: activation of alpha1-6 fucosyltransferase; Noda K et al.; The purpose of the present study was to investigate the mechanism by which nonfucosylated alpha-fetoprotein (AFP) is converted to fucosylated AFP in human hepatoma cell lines exposed to acyclic retinoid (AR), an effective drug for the secondary prevention of hepatocellular carcinoma . AR treatment (100 microM) of HepG2 and Hep3B cells significantly increased the activity and mRNA levels of alpha1-6 fucosyltransferase (alpha1-6 FucT), the enzyme responsible for the fucosylation of AFP, leading to an increase in fucosylated glycoproteins as evidenced by lectin binding measurements . Lectin immunoelectrophoresis of AFP obtained from culture media indicated that the relative percentage of nonfucosylated AFP (L1 fraction) was decreased and alpha1-6 fucosylated AFP (L3 fraction) was increased in these hepatoma cell lines after treatment with AR . The total AFP levels were, however, markedly suppressed by AR treatment, and therefore the absolute L3 fraction on the basis of the total AFP present was extremely low . These results demonstrate that AR enhances the conversion of the L1 to the L3 fraction due to the activation of alpha1-6 FucT in human hepatoma cell lines despite clinical outcome with AR treatment and the L3 fraction of AFP . Even though the dramatic decrease in AFP is the limiting factor in the synthesis of the L3 fraction and, therefore, the absolute value of fucosylated AFP is extremely low, the conversion from L1 to L3 as judged by lectin immunoelectrophoresis represents a good marker for the progress of AR treatment .

Theriogenology, 2003 Jan 1, 59(1), 189 - 205
New developments reproductive technologies in deer; Berg DK et al.; In vitro embryo production is the platform for advanced reproductive technologies, such as cloning . The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers . However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology . Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage . Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively . During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July . The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species .

Prostate Cancer Prostatic Dis, 1999 Dec, 2(5/6), 257 - 263
Isolation and partial characterization of an epithelial cell line (RPE-F344) from the regenerating prostate of a normal adult male rat; Presnell SC et al.; Normal prostate epithelial cells are difficult to propagate in vitro without experimental immortalization . The goal of this study was to isolate and characterize a propagable epithelial cell line from normal adult rat prostate . Enrichment of proliferation-competent cells was accomplished in vivo by initiating a single cycle of prostatic involution/regeneration . The RPE-F344 cell line was established from an androgen-deprived, involuted prostate four days after the initiation of regeneration by administration of testosterone . The cell line has been cultured in vitro for >50 passages, forms a uniform monolayer in culture, exhibits contact inhibition at confluence, and does not form colonies in soft agar . Immunocytochemical and RT-PCR analyses demonstrated that the RPE-F344 cells express anti-apoptotic genes associated with cell survival, and several growth factor receptors important in prostate development and homeostasis . RPE-F344 cells are p27kip1 negative, telomerase positive, and express high molecular weight cytokeratins specific for prostatic basal cells . They also express low levels of androgen receptor (AR) and prostatic acid phosphatase (PAP); features associated with secretory luminal epithelial cells . RPE-F344 cells are maintained in vitro without androgen supplementation, but addition of 15nM dihydrotesterone (DHT) to the culture media results in a significant but transient enhancement of cellular proliferation . Establishment of RPE-F344-like colonies from rat prostate is limited to the ventral and dorsal lobes of the prostate 2-4 days after initiation of regeneration, suggesting that RPE-F344 cells may originate from a stem cell-like compartment that is responsible for regenerative repopulation.

J Pharm Pharmacol, 2002 Nov, 54(11), 1529 - 34
Rapid effect of progesterone on the contraction of rat aorta in-vitro; Zhang M et al.; Progesterone induced rapid relaxation of KCl-induced contraction of rat aortic rings . The relaxant effect of progesterone on aortic rings was concentration-dependent (over the range of 10(-10) to 10(-5) M) and partially dependent on the endothelium . Application of a nitric oxide (NO) synthase antagonist N(G)-monomethyl-L-arginine (L-NMMA, 10(-5) M) after progesterone treatment partially inhibited the relaxant effects of progesterone . This suggested that part of the effect was through the production of nitric oxide . Washing out the steroid hormone in the bath solutions could quickly reverse the inhibitory effects of progesterone on phasic tension generation in aortic rings . Five minutes after washout, the tension generation in aortic rings was completely restored . Cultured endothelial cells from rat aorta increased release of NO into culture media in response to a 60-min exposure to progesterone . Aldosterone and dexamethasone were also tested, and failed to relax KCl-induced contraction of aortic rings . These data suggest that the vascular effects of progesterone are not mediated by a genomic action of this steroid, and that the vascular effects are mediated partially through endothelial NO production.

Biochem J, 2003 Mar 15, 370(Pt 3), 1097 - 109
Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury; Yonekura H et al.; The binding of advanced glycation end-products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications . In the present study, we show that the cellular constituents of small vessels, endothelial cells (EC) and pericytes express novel splice variants of RAGE mRNA coding for the isoforms that lack the N-terminal V-type immunoglobulin-like domain (N-truncated) or the C-terminal transmembrane domain (C-truncated), as well as the known full-length mRNA . The ratio of the expression of the three variants was different between EC and pericytes; the content of the C-truncated form was highest in EC, whereas the full-length form was the most abundant in pericytes . Transfection experiments with COS-7 cells demonstrated that those variant mRNAs were translated into proteins as deduced; C-truncated RAGE was efficiently secreted into the culture media, and N-truncated RAGE was located mainly on the plasma membrane . The three isoforms were also detected in primary cultured human EC and pericytes . Further, full-length and C-truncated forms of RAGE bound to an AGE-conjugated column, whereas N-truncated RAGE did not . The AGE induction of extracellular-signal-related kinase phosphorylation and vascular endothelial growth factor in EC and of the growth and cord-like structure formation of EC was abolished completely by C-truncated RAGE, indicating that this endogenous secretory receptor (endogenous secretory RAGE) is cytoprotective against AGE . The results may contribute to our understanding of the molecular basis for the diversity of cellular responses to AGE and for individual variations in the susceptibility to diabetic vascular complications.

Biol Reprod, 2003 Jan, 68(1), 60 - 6
Developmental regulation of hyaluronan-binding protein (RHAMM/IHABP) expression in early bovine embryos; Stojkovic M et al.; Hyaluronan or hyaluronic acid (HA) is a normal component of mammalian follicular, oviduct, and uterine fluids . Granulosa and expanding cumulus cells secrete large amounts of HA, and when HA is added in maturation and culture media, it improves the developmental potential of oocytes and embryos . HA regulates gene expression, signaling, proliferation, motility, adhesion, and morphogenesis . Many of these biological activities of HA are mediated through binding to the receptor for HA-mediated motility/intracellular HA-binding protein (RHAMM/IHABP) . We evaluated the presence and dynamics of RHAMM/IHABP mRNA and protein expression in different stages of in vitro-produced bovine embryos using quantitative reverse transcriptase-real time-polymerase chain reaction and immunohistochemistry . We also analyzed the effects of different culture systems on the relative abundance of RHAMM/IHABP transcripts . RHAMM/IHABP mRNA levels decreased from the 2-cell to the 16-cell stage, increased again at the morula stage, and reached their highest level at the expanded blastocyst stage . RHAMM/IHABP mRNA abundance was significantly (P < 0.05) lower in embryos recovered in serum-containing medium than in embryos from serum-free media . Immunohistochemistry revealed the presence of RHAMM/IHABP first in 8-cell stages . Whereas RHAMM staining in 8-cell and morula stages was intense, it was weaker in blastocysts . Embryonic secretion of HA increased from the 2-cell stage until the 8-cell stage and then decreased in 16-cell embryos . After this, HA secretion increased in expanded and hatched blastocyst stages . These data suggest that the positive effects of HA on in vitro-produced bovine embryos may be mediated at least in part by RHAMM/IHABP.

Virology, 2002 Nov 10, 303(1), 79 - 99
Inducible system in human hepatoma cell lines for hepatitis C virus production; Lim SP et al.; We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2 . Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening . Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation . Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment . Full-length viral plus-strand RNA was detected by Northern analyses . Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA . Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D . Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER) . Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA . Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml . Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies . Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles . Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining . Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.

Diabetes Res Clin Pract, 2003 Jan, 59(1), 25 - 30
Role of protein kinase C-angiotensin II pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels; Ikehara K et al.; The intrarenal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy . This study investigates the mechanisms for glucose-induced increase in angiotensin II (AII) production by human mesangial cells (MCs) in relation to protein kinase C (PKC) . We also examine whether locally produced AII mediates extracellular matrix protein production in high-glucose conditions . Human MCs were cultured in 5 or 33 mmol/l glucose for 8 days, and were incubated with or without 5 mmol/l GFX, a PKC inhibitor, 0.1 micromol/l candesartan cilexetil (CC), a specific type 1 AII receptor antagonist, for another 24 h . In addition, MCs grown in 5 mmol/l glucose were incubated with 0.1 micromol/l phorbol-12,13-dibutyrate (PDBu) for 24 h . AII, TGF-beta1, fibronectin and type IV collagen in the culture media were measured by ELISA . The amount of AII secreted from MCs exposed to high-glucose levels was significantly greater (P<0.01) than that in normal glucose levels . The increase in AII production was completely prevented by GFX . The addition of PDBu mimicked the effect of glucose on AII production . The glucose-induced increases in the production of TGF-beta1, fibronectin and type IV collagen were partially, but significantly restored (P<0.01) by CC, while GFX totally abolished these effects of glucose . These results suggest that elevated glucose levels stimulate AII production via mechanisms dependent on glucose-induced PKC activation in human MCs, and that locally produced AII partly mediates the increase in mesangial matrix synthesis in high-glucose conditions.

Cryobiology, 2002 Oct, 45(2), 97 - 108
Modulation of the cryopreservation cap: elevated survival with reduced dimethyl sulfoxide concentration; Baust JM et al.; The development of cryopreservation (CP) strategies has traditionally focused on the cellular chemo-osmometric characteristics attendant to the freeze-thaw process . This approach coupled with a limited understanding of cellular physiological and biochemical responses to the CP process often yields sub-optimal cell survival . Recently, we have reported on the benefits of the utilization of an intracellular-like preservation solution, HypoThermosol (HTS), as well as incorporating a molecular approach to improving CP outcome {In Vitro Cell . Dev . Biol . Anim . 36(4) (2000) 262} . We now report on the elucidation of a cryoprotective agent (CPA)-dependent survival limit (cap) associated with standard CP methodologies . We further demonstrate an elevation and shift in the CP cap through the utilization of HTS coupled with a reduction in CPA levels necessary to achieve "successful" cell preservation . METHODS: Human fibroblasts, keratinocytes, hepatic, and renal cells were cryopreserved in a standard fashion (approximately 1 degrees C min-1 cooling and storage in LN2) in culture media (serum-free) or HTS with varying levels of dimethyl sulfoxide (Me2SO) . Samples were allowed to recover for 24-h prior to survival assessment . Survival was assessed using alamarBlue (metabolic activity indicator) and calcien-AM (membrane integrity stain) in comparison with non-frozen controls . RESULTS: (1) A limit in cell survival was identified following CP in media-based CP solutions yielding a cell-type specific CPA-dependent survival limit, (2) peak cell survival resulted in the identification of "optimal" Me2SO concentrations for CP of each cell type, (3) incorporation of HTS as the carrier medium at typical Me2SO concentrations substantially elevated survival, and (4) utilization of HTS allowed for the successful preservation of all systems examined at significantly reduced Me2SO levels . CONCLUSION: The data presented in this study illustrate that the utilization of HTS as the carrier medium during CP facilitated a significant improvement in efficacy at reduced Me2SO levels . Further, the utilization of HTS offers the potential for successful Me2SO-free CP . These findings may prove significant to the advancement in the development of cell-based clinical therapies by providing an improved biocompatible CP methodology.

J Hepatol, 2003 Jan, 38(1), 76 - 83
The large GTPase dynamin is required for hepatitis B virus protein secretion from hepatocytes; Abdulkarim AS et al.; BACKGROUND/AIMS: The hepatocellular transport pathways and cellular proteins utilized during the packaging and secretion of hepatitis B virus are poorly understood . In this study, we tested if the large GTPase dynamin, a protein involved in vesicle formation and secretion at the trans-Golgi network in hepatocytes, is also used by hepatitis B virus (HBV) in secreting viral proteins . METHODS: Using HepG2.2.15 cells expressing the full-length HBV genome, we tested the effects of wild-type and mutant dynamin on the localization and secretion of two hepatitis B antigens, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) . Distribution of these two antigens was analyzed morphologically in cells transiently transfected with wild-type or mutant dynamin constructs, whereas secretion of the antigens was measured by testing for antigen levels in the media of transfected cells . RESULTS: Mutant dynamin was found to induce a striking redistribution of HBsAg and HBeAg to a perinuclear compartment, as well as a decrease in the levels of HBsAg and HBeAg present in cell culture media indicating a reduction in viral protein secretion . At the electron microscopy level, cells expressing the mutant dynamin showed a marked accumulation of viral particles in dilated cisternae of an uncharacterized cellular compartment . CONCLUSIONS: Intact dynamin function is required for secretion of HBV proteins from hepatocytes through an uncharacterized cellular compartment.

Anat Histol Embryol, 2002 Jun, 31(3), 151 - 7
Effects of bovine serum albumin and estrous cow serum on development and ultrastructure of in vitro-produced porcine embryos; Ott M et al.; This study evaluated the effects of bovine serum albumin (BSA; 4 mg/ml) and estrous cow serum (ECS; 10%) in North Carolina State University (NCSU) 23 medium on the development of in vitro-matured and in vitro-fertilized porcine oocytes . Early cleavage rate was significantly (P < 0.05) higher in NCSU/ECS (71.3 +/- 14.7%) vs . NCSU/BSA (60.6 +/- 4.7%) . Cleavage beyond the four-cell stage was not different between the two culture media (43.5 +/- 9.5% and 41.4 +/- 17.7%, respectively) . The proportion of development to blastocysts was--with borderline significance (P = 0.05)--higher in NCSU/BSA (28.0 +/- 4.4%) than in NCSU/ECS (20.4 +/- 7.3%) . Blastocysts produced in NCSU/BSA had significantly (P < 0.001) higher cell numbers than those cultured in NCSU/ECS (29.5 +/- 20.1 vs . 16.9 +/- 10.8) . The ultrastructure of in vitro-produced blastocysts from both culture systems was compared vs . in vivo-derived blastocysts . The latter showed a clear differentiation between trophectoderm (TE) and inner cell mass (ICM) cells . The TE cells were anchored to other TE cells or ICM cells by long, well-developed junctional complexes . The apical membrane of trophoblast cells was covered with numerous microvilli . Mitochondria were abundant, round to elongated in shape, and showed clear transverse cristae . The ultrastructure of blastocysts cultured in NCSU/BSA mimicked that of in vivo-derived embryos closely . In contrast, blastocysts from the NCSU/ECS culture system displayed an irregular ultrastructure with reduced numbers of organelles and numerous cytoplasmic inclusions, such as lipid-yolk-vacuoles and vacuoles with lipid content . In some sections of these embryos, cellular debris was detected in cytoplasm . The shape of mitochondria was more ovoid and cristae were not visible . In summary, our results demonstrate a beneficial influence of ECS in the culture medium on initial cleavage of in vitro-produced porcine embryos . Clearly negative effects of ECS in the subsequent culture period are associated with marked ultrastructural changes of embryonic cells.

J Virol, 2003 Jan, 77(1), 416 - 22
Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation; Ponsel D et al.; The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published . We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis . For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues . The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions . Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions . Twenty-seven mutations were compatible with virion formation . Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike . Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels . These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell . These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.

Fertil Steril, 2002 Dec, 78(6), 1254 - 60
Improvement in early human embryo development using new formulation sequential stage-specific culture media; Cooke S et al.; OBJECTIVE: To determine whether altering selected components of sequential culture media can improve early development variables of human embryos . DESIGN: Prospective, randomized, sibling oocyte split trial . SETTING: Private ART center . PATIENT(S): Two hundred eight undergoing treatment with in vitro fertilization or microinjection . INTERVENTION(S): Oocytes from each patient were randomly allocated to fertilization and cleavage media of a control and a trial culture medium formulation . MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, and uncontrolled division; average embryo morphology score; blastomeres per embryo; embryo score parameter (number of blastomeres x embryo morphology grade); and embryo utilization.The trial media resulted in a higher fertilization rate, higher cleavage rate, lower rate of uncontrolled division, higher number of blastomeres per embryo, higher average embryo morphology score, a higher embryo score parameter, and higher embryo utilization rate compared to the control media . All differences were statistically significant . CONCLUSION(S): Improved sequential stage-specific culture media can reduce the occurrence of severe human embryo fragmentation and improve developmental variables in early IVF- and ICSI-generated embryos.

Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 17107 - 12 Epub 2002 Dec 10.
Protection against ischemic brain injury by protein therapeutics; Asoh S et al.; Preventing massive cell death is an important therapeutic strategy for various injuries and disorders . Protein therapeutics have the advantage of delivering proteins in a short period . We have engineered the antiapoptotic bcl-x gene to generate the super antiapoptotic factor, FNK, with a more powerful cytoprotective activity . In this study, we fused the protein transduction domain (PTD) of the HIVTat protein to FNK and used the construct in an animal model of ischemic brain injury . When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 h . It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively . The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM . Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action . Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected i.p . into mice can have access into brain neurons . When injected i.p . into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia . These results suggest that PTD-FNK has a potential for clinical utility as a protein therapeutic strategy to prevent cell death in the brain.

Mutagenesis, 2003 Jan, 18(1), 81 - 6
A comparison of folic acid and 5-methyltetrahydrofolate for prevention of DNA damage and cell death in human lymphocytes in vitro; Wang X et al.; Folic acid (FA), the most oxidized and stable form of folate, is commonly used as a dietary supplement and in culture media . FA must be reduced and methylated to become the metabolically active form found in blood and utilized by tissues, i.e . 5-methyltetrahydrofolate (5-MeTHF) . 5-MeTHF is the methyl group donor required for the conversion of homocysteine to methionine catalyzed by vitamin B(12)-dependent methionine synthase . It is hypothesized that 5-MeTHF may be more effective than FA in reducing spontaneous DNA damage and improving cell proliferation because, unlike FA, it can donate a methyl group for methionine synthesis, which is required for cell division via polyamine production and for maintenance methylation of DNA after its conversion to S-adenosylmethionine . We aimed to determine whether FA and 5-MeTHF differed in their capacity to prevent genetic damage and cell proliferation of human lymphocytes in vitro . Lymphocytes from eight female volunteers (40-48 years) were cultured in RPMI 1640 medium containing 12-120 nM FA or 5-MeTHF for 9 days . Mitogenesis was stimulated with phytohemagglutinin and the medium changed on days 3 and 6 . Cytokinesis was inhibited by adding cytochalasin B on day 8 and cells were harvested and transferred to microscope slides on day 9 . Chromosome damage, cell death and cytostasis was measured using the cytokinesis-block micronucleus assay in its comprehensive mode . The results showed that the frequency of micronucleated binucleate cells was significantly lower at 120 nM FA compared with 120 nM 5-MeTHF (P < 0.05), however, at 12 nM concentration both forms of folate were associated with increased frequency of micronuclei and nuclear buds relative to 120 nM (P < 0.05) . Apoptosis tended to be significantly higher in 5-MeTHF cultures compared with FA cultures, however, necrosis and nuclear division were similar between cultures . We conclude that 5-MeTHF is not more efficient than FA in preventing human lymphocyte genomic instability in this in vitro system . Further research is needed to clarify the role of choline and methionine concentration and the importance of the reduced folate carrier and the folate receptor in determining the relative bioavailability of 5-MeTHF and FA with regard to genome stability.

Br J Haematol, 2002 Dec, 119(4), 1052 - 8
Two double heterozygous mutations in the F7 gene show different manifestations; Nagaizumi K et al.; We sequenced the factor VII gene (F7) in two unrelated Japanese patients with factor VII (FVII) deficiency . In the first (an asymptomatic 46-year-old man with FVII activity and antigen levels of 1.2% and 21% of normal respectively), novel E25K and H348Q mutations were identified in the doubly heterozygous state . In transiently transfected HEK293 cells, the level of FVII-E25K mutant activity in the culture media was significantly lower than that of FVII wild type, whereas the antigen levels of both proteins were similar . This suggests that the E25K mutation is associated with a dysfunctional FVII molecule . In the second patient (a 47-year-old woman with FVII activity and antigen levels of less than 1% and 6% respectively), an IVS4+1 mutation and a novel -96C to T transition were detected in the double heterozygous state . In electrophoretic mobility shift assays, the -96T mutation was shown to disrupt binding of Sp1.

J Orthop Res, 2002 Nov, 20(6), 1246 - 52
Prostaglandin E1 analog inhibits the microglia function: suppression of lipopolysaccharide-induced nitric oxide and TNF-alpha release; Chuai M et al.; Release of nitric oxide and TNF-alpha, a toxic cytokine, have been reported to accelerate neuronal damage under several pathological conditions, such as trauma or ischemia in the central nervous system . In the present study, we tested the effect of alprostadil alfadex, a prostaglandin E1 analog, on cultured microglia from the rat spinal cord . The cultured microglia were exposed to lipopolysaccharide (LPS) (100 ng/ml), an endotoxin, for 24 h, then the released nitric oxide and TNF-alpha in the culture media was analyzed . The released nitric oxide was detected by the Griess reaction and released TNF-alpha was measured using ELISA method . The LPS-induced nitric oxide release was inhibited by the simultaneous addition of alprostadil alfadex in a dose-dependent manner (0.1-100 microM) . The LPS-induced TNF-alpha release was also inhibited by alprostadil alfadex addition (0.1-100 microM) . The IC50 values of alprostadil alfadex on nitric oxide and TNF-alpha release were about 1 and 10 microM, respectively . These results suggest that prostaglandin E1 possibly protects spinal cord neurons from several types of neurodegenerative damage, not only via increased blood supply, but also via inhibition of pathological immunoreactions of activated microglia.

Reprod Biomed Online, 2002 Jul-Aug, 5(1), 39 - 42
Energy substrates, mitochondrial membrane potential and human preimplantation embryo division; Wilding M et al.; Carbohydrate additives to modern embryo culture media are based on three basic energy sources, glucose, pyruvate and lactate . Although the use of these substrates is almost universal, debate continues as to the roles of the individual components in the human . This is mainly due to the lack of human embryos for research and the reliance on animal model systems . In the present work, the human embryo was used to study the role of the above simple substrates in the maintenance of the mitochondrial membrane potential and cell division . The mitochondrial membrane potential was measured with fluorescence techniques . Cell division was scored as the number of blastomeres on day 3 . Both the mitochondrial membrane potential and cell division were dramatically lost in the absence of energy sources . The mitochondrial membrane potential and cell division were normal in media containing all three energy sources, or in pyruvate-containing media . Both glucose and lactate individually proved poor energy sources for the maintenance of the mitochondrial membrane potential . However, cell division continued in the presence of glucose, suggesting that some energy production can continue . These data suggest that pyruvate is an absolute requirement for mitochondrial respiration and cell cleavage during human preimplantation development . The role of lactate is as yet unclear.

Hybrid Hybridomics, 2002 Oct, 21(5), 321 - 31
Protective, anti-tumor monoclonal antibody recognizes a conformational epitope similar to melibiose at the surface of invasive murine melanoma cells; Dobroff AS et al.; Polyclonal and monoclonal antibodies (MAbs) have been raised against B16F10 cells collected from growing tumors in vivo or grown in culture media supplemented with normal mouse serum to avoid xenogeneic reactivity . Antibody binding to glutaraldehyde-fixed melanoma cells and Melan A melanocytes was assayed using chemiluminescent-enzyme-linked immunosorbent assay (CL-ELISA) for increased sensitivity . Most of the reactivity of antitumor polyclonal IgG (92%) was inhibitable by a carbohydrate pool consisting of melibiose, mannose, lactose, and sialic acid . Two monoclonal IgG(2a) antibodies, A4 and B11, had their reactivity to melanoma cells completely and specifically inhibited by melibiose . MAb A4 did not bind to alpha-galactosyl residues abundantly expressed in a protozoan mucin used as substrate, and its binding to the tumor cells was not affected by alpha-galactosidase treatment or addition of alpha-methyl-galactopyranoside or raffinose . Recognition of a mimotope similar to melibiose is suggested . MAb is cytotoxic in vitro in a complement-mediated reaction and effectively neutralizes melanoma cells protecting syngeneic mice against tumor development in vivo . This MAb is thus an important tool for further studies on antitumor adjuvant therapy combined with other agents associated with immuno- and chemotherapy of invasive melanoma.

J Clin Laser Med Surg, 2002 Oct, 20(5), 241 - 4
Light-induced replication of nanobacteria: a preliminary report; Sommer AP et al.; OBJECTIVE: The purpose of the present study was to investigate the effect of light on nanobacteria . BACKGROUND DATA: Since their first description in literature, it is not clear whether the nanoparticles called "nanobacteria" are alive or not . The 80-1,000-nm-sized spherical particles are protected by a crystalline carbonate apatite shell and are culturable in cell culture media . Present in mammalians, including humans, nanobacteria seem to cause diseases related to biomineralization processes . Mesoscopic structures found on Martian meteorites and terrestrial rocks indicated that nanobacteria-like biological objects forming apatite, a material fairly transparent to visible light, could have been present on the primitive Earth during an era with the sun as the principal terrestrial energy source . MATERIALS AND METHODS: To evaluate possible biomedical effects of therapeutically relevant irradiation sources on nanobacteria, we irradiated nanobacteria cultures with polarized light and laser-light at low, nonthermal energy density levels . RESULTS: Our observations indicated that nanobacteria are alive . Polarized white light was found to clearly accelerate their replication in vitro, resulting in significant dose-dependent increases in the turbidity of the cultures, compared to nonirradiated controls . Laser irradiation did not affect their replication . CONCLUSION: The possibility that primordial and present nanobacteria could have been not only exposed to, but actively harvested, solar irradiation for their own development suggests itself . Considering that there exists no published material on the action of light on nanobacteria, the reported effects are expected to have an impact on modeling biomineralization processes, associated photoreceptor mechanisms, and astrobiological and evolutionary theories-on Earth and in space.

J Nutr, 2002 Dec, 132(12), 3754 - 9
Lycopene inhibits proliferation and enhances gap-junction communication of KB-1 human oral tumor cells; Livny O et al.; Cell-cell interaction via gap junctions is considered to be a key factor in tissue homeostasis, and its alteration is associated with the neoplastic phenotype . Experimental and epidemiologic data suggest that carotenoids, particularly lycopene and beta-carotene, can reduce the risk of certain cancers . The aim of this study was to assess whether lycopene and beta-carotene interfere at some stage with the carcinogenic processes in human cancer cells derived from the oral cavity . KB-1 cells, originating from a human oral cavity tumor, were incubated with different concentrations of lycopene or beta-carotene delivered via the cell culture media from stock solutions in tetrahydrofuran . Lycopene strongly and dose dependently inhibited proliferation of KB-1 human oral tumor cells . beta-Carotene was a far less effective growth inhibitor . Lycopene (3 and 7 micro mol/L) significantly upregulated both the transcription (P < 0.005) and the expression (P < 0.05) of connexin 43, a key protein in the formation of gap-junctional communication . beta-Carotene (3 micro mol/L) tended to upregulate connexin 43 expression (P = 0.07) and significantly affected transcription of connexin 43 at 7 micro mol/L (P < 0.05) . Gap-junctional communication measured by scrape-loading dye transfer and electron microscopy showed that lycopene enhanced gap-junctional communication between the cancer cells, whereas beta-carotene was less effective in this regard . The pattern of cellular uptake and incorporation into cancer KB-1 cells differed significantly between the carotenoids . beta-Carotene was avidly and rapidly incorporated into KB-1 cells, whereas lycopene uptake into the cells took place after longer incubation periods and only at the highest concentrations . The results of the present study further support the hypothesis that carotenoids in general, and lycopene in particular, may be effective anticarcinogenic agents in oral carcinogenesis.

Reprod Fertil Dev, 2002, 14(5-6), 275 - 86
In vitro production of pig embryos: a point of view; Coy P et al.; Porcine embryos have become raw materials for different programmes of reproductive biotechnology and the in vitro production of embryos has some advantages over in vivo production in gene transfer programmes and for xenotransplantation . Despite this promising future, several problems limit the success of the in vitro production (IVP) of viable porcine embryos . Porcine IVP has not been fully developed because of several problems associated with different techniques, such as incomplete final maturation status after in vitro maturation, a high incidence of polyspermy after in vitro fertilization and a low development rate and poor quality of blastocysts at the end of culture . The results could be improved with studies comparing in vivo and in vitro conditions, standardization of techniques for sperm processing, testing new additives in the culture media and developing intracytoplasmic sperm injection procedures . The first objective of the present article is to summarize the main studies published on the subject . Second, we provide a guide for researchers starting work on the IVP of pig embryos, making special mention of first papers and the most recent achievements for each of the different techniques . Third, we provide suggestions for future experiments designed to improve the results of each technique.

Brain Res Bull, 2003 Jan 15, 59(4), 315 - 8
Catalase prevents elevation of {Ca(2+)}(i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology; Li W et al.; Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS) . The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ({Ca(2+)}(i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury . The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of {Ca(2+)}(i) . Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of {Ca(2+)}(i) after 1-3 days . Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in {Ca(2+)}(i) (p<0.01) . Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of {Ca(2+)}(i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of {Ca(2+)}(i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner . Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the {Ca(2+)}(i) increases caused by ethanol in the first 24h . Catalase at a concentration of 1000 units/ml inhibited completely excessive {Ca(2+)}(i) accumulation . The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.

Reprod Domest Anim, 2002 Dec, 37(6), 362 - 8
Effects of VEGF and bFGF on proliferation and production of steroids and nitric oxide in porcine granulosa cells; Grasselli F et al.; Ovarian angiogenesis, which is currently considered to be of crucial importance in controlling the growth of developing follicles, is a physiological process driven by a variety of angiogenic factors . Among these, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been recognized as key players in promoting cell growth and differentiation . Porcine granulosa cells from small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles were seeded at different densities in DMEM:Ham's F12 (1:1) with or without different concentrations of VEGF or bFGF . After 48 h of culture, media were assayed for oestradiol (E2) 17beta, progesterone (P4), nitric oxide (NO) and VEGF levels; in addition, cell proliferation was evaluated by 3H-thymidine incorporation assay . Both bFGF and VEGF effects on E2 and P4 production by cultured granulosa cells resulted to be dependent on follicle size . The bFGF was always ineffective in modulating cell proliferation, while VEGF exerted an inhibitory effect on the proliferation in the small follicle group and a stimulatory one in the medium and large follicle groups . The bFGF consistently reduced NO levels in culture media . The VEGF appeared to be ineffective in modifying NO production in the small follicle group, while it was stimulatory in the medium follicle group and inhibitory in the large follicle group . Basal VEGF production was higher in cells from the large follicle as compared with the small and medium follicle groups, and it was unaffected by bFGF . These results suggest that VEGF plays a modulatory role in granulosa cell functional activity and it is possibly involved in the regulation of follicle growth; on the contrary, bFGF does not appear to represent a significant regulatory factor in our cellular model, except for an inhibitory action on the production of NO, whose anti-angiogenic properties need to be further substantiated.

Reprod Domest Anim, 2002 Dec, 37(6), 352 - 6
Effects of serum-free culture media on in vitro development of domestic cat embryos following in vitro maturation and fertilization; Murakami M et al.; This study was conducted to determine the adequate medium for a serum-free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF) . Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h . After insemination, the oocytes were cultured in five media {Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK-1) and CR1aa}, each of which contained 0.4% bovine serum albumin . There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF . The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK-1, TCM199 and CR1aa . Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage . There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK-1, TCM199 and CR1aa . These results indicate that the type of serum-free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK-1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum-free culture system.

Reprod Domest Anim, 2002 Dec, 37(6), 347 - 51
Effect of albumin and polyvinyl alcohol on the vitality, motility and acrosomal integrity of canine spermatozoa incubated in vitro; Risopatron J et al.; Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa . The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM-199), sperm culture medium (Sp-TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa . Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim-up technique . Sperm progressive motility was assessed using a phase contrast microscope . Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa-Trypan blue) . The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules . A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%) . The percentage of acrosome-intact spermatozoa was markedly higher after HTF (94.1%) than after TCM-199 (70.1%) or Sp-TALP (71.0%) without supplementation . It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.

BioDrugs, 2002, 16(6), 389 - 401
Stem cells for neurodegenerative disorders: where can we go from here?
Le Belle JE, Svendsen CN.
The use of stem cells in cell replacement therapy for neurodegenerative diseases has received a great deal of scientific and public interest in recent years . This is due to the remarkable pace at which paradigm-changing discoveries have been made regarding the neurogenic potential of embryonic, fetal, and adult cells . Over the last decade, clinical fetal tissue transplants have demonstrated that dopaminergic neurons can survive long term and provide functional clinical benefits for patients with Parkinson's disease . Pluripotent embryonic stem cells and multipotent neural stem cells may provide renewable sources that could replace these primary fetal grafts . Considerable advancement has been made in generating cultures with high numbers of neurons in general and of dopaminergic neurons using a varied array of techniques . However, much of this encouraging progress still remains to be tested on long-term expanded human cultures . Further problems include the low survival rate of these cells following transplantation and the tumorigenic tendencies of embryo-derived cells . However, pre-differentiation or genetic modification of stem cell cultures prior to transplantation may help lead to the generation of high numbers of cells of the desired phenotype following grafting . Boosting particular factors or substrates in the culture media may also protect grafted neurons from oxidative and metabolic stress, and provide epigenetic trophic support . Possible endogenous sources of cells for brain repair include the transdifferentiation of various types of adult cells into neurons . Despite the excitement generated by examples of this phenomenon, further work is needed in order to identify the precise instructive cues that generate neural cells from many other tissue types, and whether or not the new cells are functionally normal . Furthermore, issues such as cell homogeneity and fusion need to be addressed further before the true potential of transdifferentiation can be known . Endogenous stem cells also reside in the neurogenic zones of the adult brain (ventricle lining and hippocampus) . Further elucidation of the mechanisms that stimulate cell division and migration are required in order to learn how to amplify the small amount of new cells generated by the adult brain and to direct these cells to areas of injury or degeneration . Finally, a more fundamental understanding of brain injury and disease is required in order to circumvent local brain environmental restrictions on endogenous cell differentiation and survival.

Med Mycol, 2002 Oct, 40(5), 447 - 54
Production of culture filtrates of Sporothrix schenckii in diverse culture media; Mendoza M et al.; Mycelial-form Sporothrix schenckii was studied to determine if growth in complex (Sabouraud-dextrose) or defined culture media (minimal medium, Gibco Medium 199 with and without Hepes buffer) with differing initial pH levels would affect expression of antigen components . Cultures were evaluated by continuous monitoring and serial sampling for various parameters . Great variation was seen in the protein and antigenic patterns induced by the different media . The expression of a 55 kDa component accompanied by significant acidification of the culture medium at the beginning of the stationary growth phase was notable in Sabouraud medium . In the chemically defined media, a 90 kDa component was expressed that reacted with sera from patients with sporotrichosis . The pH in these media showed little change during the different growth phases of the fungus . Among the media studied, minimal medium favored the expression of the greatest number of antigenic components . In all of the assays, the stationary growth phase appeared to be optimal for content of antigenic components . Cross reactions were not observed with any of the culture filtrates using sera from patients with other mycoses.

Tissue Eng, 2002 Oct, 8(5), 739 - 51
Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction; Lodie TA et al.; Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media . Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential . In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days . Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone . Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method . CD44 expression was variable, apparently dependent on serum concentration . Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively . On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.

Hum Reprod, 2002 Dec, 17(12), 3153 - 6
A modified RT-PCR technique to screen for viral RNA in the semen of hepatitis C virus-positive men; Cassuto NG et al.; BACKGROUND: Our objective was to use an adapted RT-PCR technique to assess the presence of hepatitis C virus (HCV) in semen and also in different density gradient semen fractions collected from men with chronic viral hepatitis participating in an assisted reproduction programme . METHODS: This study included 50 semen samples from 35 HCV(+) men, with active viral replication assessed by RT-PCR, collected the day of oocyte retrieval and used for assisted reproduction . These samples were subjected to standard assisted reproduction sperm preparation conditions, using density-gradient centrifugation with 45 and 90% layers . Aliquots of semen, 45 and 90% fractions, and embryo culture media were frozen at -80 degrees C for subsequent virological analyses . All aliquots were tested with a commercially available HCV RNA assay, adapted for use with semen after a number of technical changes . This assay yielded a sensitivity of 50-100 HCV RNA copies/ml and strongly diminished the effect of seminal amplification inhibitors . RESULTS: HCV RNA was detected in 7/50 (14%) semen samples tested, 5/35 (14.3%) men . HCV RNA was found in only 1/50 45% fractions but never in the 90% fraction or embryo culture media . Sera from 3/5 men contained 3.19-7.40 x 10(5) IU/ml, while the two others had 4.5 and 11.7 x 10(6) IU/ml . However, HCV RNA was quantified at <600 IU/ml in the HCV(+) semen of these five patients . The ongoing pregnancy rate was of 20% (10/50) with one delivery at the time of the present report . No anti-HCV antibody was found in any of the women or the newborn . CONCLUSIONS: Although HCV is present at low concentrations in the semen of a few HCV(+) patients, no purified sperm fraction (i.e . 90% fraction) used in assisted reproduction was HCV(+) and no seroconversion was observed in the women and the newborn, thereby suggesting a very low risk of virus transmission . Nevertheless, because the presence of HCV in semen implies a possible risk of nosocomial contamination, safety regulations must be strictly applied in assisted reproduction laboratories.

Tsitologiia, 2002, 44(7), 702 - 11
{Random distribution of the level of growth factors in malignant and revertant clones from murine and human tumors}; Lavrovskii VA et al.; It is well known that artificial increase in expression of growth factors and their receptors can lead to tumorigenic transformation of cells . Additionally, multiple data on the increased expression of growth factors in many human and animal tumorigenic cells have been published . Nevertheless description of the functional role of endogenous growth factors in maintenance of tumorigenic phenotype remains obscure . Previously, we described a new model for studying neoplastic transformation and dormant metastasis . This model consists of cognate tumorigenic and nontumorigenic cell clones, the latter being obtained as a result of spontaneous reversion of tumorigenic ones . All revertant clones demonstrate the three well known features of normal cells: monolayer growth on a plastic, incapability to grow in soft agar with regular culture media with 10% FCS, and incapability to form tumors in syngeneic animals . Using RT-PCR, we measured expression ratios of main growth factors, commonly believed to be associated with malignization, in tumorigenic and revertant clones of our model . For some of these clones, we also measured an activity of growth factors in conditioned media . The data obtained argue that the levels of growth factor expression, measured by both the methods, are distributed between tumorigenic and revertant clones in a sporadic manner and do not correlate with cell tumorigenicity . Thus, our experimental observations enable us to consider the variability of growth factor expression as insignificant events in the reversion of tumorigenic cells to a nontumorigenic phenotype.

J Agric Food Chem, 2002 Dec 4, 50(25), 7220 - 5
Antioxidant and prooxidant effects of phenolics on pancreatic beta-cells in vitro; Lapidot T et al.; A number of natural phenolic compounds display antioxidant and cell protective effects in cell culture models, yet in some studies show prooxidant and cytotoxic effects . Pancreatic beta-cells have been reported to exhibit particular sensitivity to oxidative stress, a factor that may contribute to the impaired beta-cell function characteristic of diabetes . The aim of this study was to examine the potential of natural phenolics to protect cultured pancreatic beta-cells (betaTC1 and HIT) from H(2)O(2) oxidative stress . Exposure of cells to H(2)O(2) led to significant proliferation inhibition . Contrary to what one should expect, simultaneous exposure to H(2)O(2) and the phenolics, quercetin (10-100 microM), catechin (50-500 microM), or ascorbic acid (100-1000 microM), led to amplification of proliferation inhibition . At higher concentrations, these compounds inhibited proliferation, even in the absence of added H(2)O(2) . This prooxidant effect is attributable to the generation of H(2)O(2) through interaction of the added phenolic compounds with as yet undefined componenets of the culture media . On the other hand, inclusion of metmyoglobin (30 microM) in the culture medium significantly reduced the prooxidant impact of the phenolics . Under these conditions, quercetin and catechin significantly protected the cells against oxidative stress when these components were present during the stress period . Furthermore, significant cell protection was observed upon preincubation of cells with chrysin, quercetin, catechin, or caffeic acid (50 microM, each) prior to application of oxidative stress . It is concluded that provided artifactual prooxidant effects are avoided, preincubation of beta-cells with relatively hydrophobic natural phenolics can confer protection against oxidative stress.

Can J Physiol Pharmacol, 2002 Oct, 80(10), 1030 - 3
Peripheral blood mononuclear cell production of TNF-alpha in response to North American ginseng stimulation; Zhou DL et al.; North American ginseng (Panax quinquefolium) root extract (NAGE) with known ginsenosides composition was examined for its affinity to stimulate human tumour necrosis factor alpha (TNF-alpha) production in human peripheral blood mononuclear cells . Case studies were conducted in three donors, one that was diagnosed with an atopic allergy and two that were normal, healthy subjects . Cultured mononuclear cells were incubated with varying concentrations of NAGE for up to 72 h and culture media were tested for TNF-alpha concentration . Direct stimulation of mononuclear cell TNF-alpha production in vitro by NAGE occurred as early as 6 h with 200 microg NAGE/mL . The stimulation of TNF-alpha production was confirmed by TNF-alpha mRNA gene expression . These interesting results show the immunostimulating activity of NAGE components in reference to TNF-alpha production . This observation requires further investigation with more subjects to determine the affinity of ginseng in stimulating the human immune system . Moreover, the method of evaluating this response is very useful for standardizing ginseng extracts to a known bioactivity.

Hepatology, 2002 Dec, 36(6), 1488 - 97
Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells; Yasuchika K et al.; Because of a donor shortage problem in liver transplantation, cell transplantation has been anticipated as a useful bridge or substitute therapy, and has necessitated the development of cell sources other than donated organs . Therefore, the use of fetal hepatic progenitor cells (HPCs) is now being focused on . In this study, we intended to establish an efficient ex vivo nonviral gene-transfer system using a newly developed isolation and culture system for mouse fetal HPCs . Fetal HPCs, characterized using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for lineage markers, were collected from E13.5 Balb/c mice using change in size because of cell aggregation by their homophilic cell-to-cell binding occurring during suspension culture . Optimal conditions for culture and ex vivo gene transfection for fetal HPCs were determined by (3)H-thymidine incorporation and the expression efficacy of transfected red fluorescent protein (DsRed) gene in different culture media . The optimum timing for gene transfection was also evaluated . To evaluate the in vivo expression of the transferred gene, DsRed-transferred fetal HPCs were transplanted into 70% partially hepatectomized allogenic mice . The highest efficacy of DsRed gene transfection into fetal HPCs in vitro (45% +/- 12.3%) was achieved with culture media, which also enabled the highest (3)H-thymidine incorporation, containing the deleted form of hepatocyte growth factor (dHGF) and insulin, and when transfection was performed immediately after isolation . In vivo DsRed expression in fetal HPCs was maintained concomitantly with albumin expression even after HPC transplantation . In conclusion, we established a highly efficient in vitro gene transfer system for mouse fetal HPCs using a newly developed isolation and culture system.

Biol Reprod, 2002 Dec, 67(6), 1952 - 8
Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system; Arechavaleta-Velasco F et al.; The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes . Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture . These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies . Levels of proMMP-9 in culture media were evaluated by zymography . Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation . ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta . The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta . No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS . In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS . Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS . Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion . Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.

Biol Reprod, 2002 Dec, 67(6), 1726 - 33
Mouse ovarian follicles secrete factors affecting the growth and development of like-sized ovarian follicles in vitro; Spears N et al.; A series of experiments have been carried out to determine whether follicles secrete factors able to affect the growth and development of other, like-sized follicles . Late preantral mouse ovarian follicles were either cocultured or cultured in media conditioned by previously cultured follicles . In particular, the experiments examined whether follicles do secrete such factors, whether the level of FSH in the culture media can affect that process, and what the nature of such secretory factor(s) might be . First, pairs of follicles were cocultured across a polycarbonate membrane containing pores . This showed that communication between the follicles resulted in the stimulation of growth and that the stimulation was due, at least in part, to the production of secretory factor(s) . In subsequent experiments, follicles were cultured in media that had been preconditioned by previously cultured follicles . The concentration of FSH in the cultures determined the effect of the conditioned media: conditioned media was stimulatory to follicle growth when levels of FSH remained high throughout the culture, but inhibitory when FSH levels were dropped midway through the cultures . Heat inactivation removed this inhibitory effect, showing that the factor was likely to be a protein; addition of follistatin to the conditioned media did not alter its effect, indicating that the factor was unlikely to be activin . We have shown through a series of culture experiments that mouse follicles secrete factor(s) that can affect the development of other like-sized follicles when cultured from the late preantral to Graafian stages . Furthermore, we have shown that the effect (or production) of such factors is dependent on the FSH environment of the follicles.

Chin J Traumatol, 2002 Dec, 5(6), 374 - 9
Osteogenic potential of rabbit marrow stromal stem cells cultured in vitro: a histochemical and scanning electron microscopic study; Wan C et al.; OBJECTIVE: To further investigate the osteogenic potential of rabbit marrow stromal stem cells cultured in vitro . METHODS: Rabbit marrow stromal stem cells were isolated by density gradient centrifugation method and amplified in the flasks, using the osteogenic inducing conditions (OGC) as the culture media . The osteogenic potential of marrow stromal stem cells were investigated by means of bone-seeking fluorescence (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue-Sirius red (AS) staining, and scanning electron microscope . RESULTS: After being passaged, the marrow stromal stem cells increased in number, became confluent and formed multi-layer structure . The stromal stem cells excreted innumerable tiny granules, heaping up on the cell body and merging gradually into foggy substances . These foggy substances kept on enlarging and formed round, oval, or flake-like nodules . These nodules revealed bright golden yellow fluorescence under fluorescence microscope when labelled with tetracycline . Histochemical study with specific new bone staining with ARS revealed positive calcium reaction, both denoting that they were newly formed bone tissues . After they were stained with AS, collagen and acid mucopolysaccharide were shown . Under scanning electron microscope, three types of cells with different configurations were found . They were globular cells, spindle-shaped cells and polygonal or polygonal cells . Granules were excreted from the cells and heaped up on the cell body . Needle-shaped and irregularly rectangular crystals also appeared and agglomerated with the granules to form nodules and trabecula-like or flake-like structures . CONCLUSIONS: Sequence of events of bone formation by rabbit marrow stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast differentiation from marrow stromal stem cells and the possible application in orthopaedics.

Proteomics, 2002 Nov, 2(11), 1601 - 15
Proteomic analysis of secreted muscle components: search for factors involved in neuromuscular synapse formation; Gajendran N et al.; Denervated but not innervated skeletal muscles secrete polypeptides that are involved in neuromuscular synapse formation . With the aim of identifying such components, metabolically labeled polypeptides in extracts from denervated and innervated muscles were submitted to two-dimensional gel electrophoresis, and the abundance of individual molecular species was compared . Consistent differences between the proteomic maps from the two sources of muscles were seen . Likewise, proteomic maps of polypeptides from organ culture media conditioned by chronically denervated muscles and by control muscles revealed consistent differences, but the abundance of material within individual spots from conditioned media was not sufficient for analysis by mass spectrometry . Since it was not possible to match the patterns from muscle extracts and from conditioned media, it has been established that extract of Sol8 muscle cells was a satisfactory source of material for analysis . From 1,200 spots identified on the proteomic map from Sol8 cells by image analysis, some 140 have been defined by mass spectrometric analysis . In order to identify the components that are shared by secreted molecules from denervated muscles and Sol8 cells, a mixture of extracts from the two sources was co-electrophoresed and a shared proteomic pattern was established by visualization of metabolically labeled spots from the conditioned medium and of silver stained spots from the Sol8 cells . More than 100 spots sharing x/y coordinate localization could be seen on the pattern . Of these, fourteen were among those identified by mass spectrometry . It is concluded that co-electrophoresis of radioactively labeled polypeptides from conditioned media with extracts from Sol8 cells can be used to mark in the proteome of Sol8 cells those polypeptides that are secreted at low abundance by adult muscles . Their higher abundance in Sol8 cells opens the possibility for further scrutiny of spots by mass spectrometry or by microsequencing.

Exp Cell Res, 2002 Nov 15, 281(1), 128 - 39
Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression; Sommi P et al.; Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer . In vitro studies have shown that H . pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones . The mechanisms by which H . pylori promotes perturbation of cell proliferation are not yet elucidated . To investigate whether products released by H . pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H . pylori broth culture filtrate . Cell cycle analysis showed that a H . pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease . The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity . In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H . pylori-released factor(s) . In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein . A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa . These results suggest that H . pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1) . This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.

J Orthop Trauma, 2002 Nov-Dec, 16(10), 717 - 22
Bone formation following intramedullary femoral reaming is decreased by indomethacin and antibodies to insulin-like growth factors; Bhandari M et al.; OBJECTIVE: We aimed to: 1) . compare rates of in vitro bone formation following reamed and nonreamed intramedullary fixation in a murine model of femoral fracture healing; and 2) . examine whether antibodies to insulin-like growth factor (IGF) I, IGF II, or indomethacin (an inhibitor of the inflammatory process) affect bone formation following intramedullary reaming . DESIGN: Experimental study . PARTICIPANTS: Twenty-four C57 black mice were randomized to two groups: reamed ( = 12), and nonreamed intramedullary nail insertion ( = 12) . INTERVENTION: In the reamed group, the femoral canals were successively reamed with 30-, 27-, 25-, and 23-gauge stainless steel pins and stabilized with a 27-gauge pin . In mice randomized to the nonreamed group, a 27-gauge pin was inserted . An external three-point bending force created a midshaft transverse femoral fracture . Seven days postsurgery, each mouse was killed, and the right femur was removed . Following pin removal, the callus was minced, the bone marrow was removed, and both were ultracentrifuged at 1200 rpm for 5 minutes . The supernatent was cocultured with 3-day-old murine calvarial cells in culture media . At day 5 of culture, reamed plasma and calvarial cell cocultures were exposed to either 1.0 micro g/mL of anti-IGF I, 1.0 micro g/mL of anti-IGF II, 2 micro M indomethacin, or served as controls (calvarial cells only) . The cells were cultured for a total of 21 days . MAIN OUTCOME MEASUREMENTS: The number of bone nodules was quantified by light microscopy . RESULTS: Reamed pin insertion resulted in 4.1-fold and 8.9-fold increases in the mean number of bone nodules compared to pins inserted without reaming and controls, respectively (399 +/- 40.0 vs . 97.0 +/- 21.0, < 0.001) . The positive effect of intramedullary reaming on bone nodule formation was reversed with the administration of antibodies to IGF I and IGF II . The addition of anti-IGF I or anti-IGF II to calvarial, or osteoblastlike, cells treated with supernatent from the callus and bone marrow of mice with prior intramedullary reaming resulted in significant declines in the mean number of bone nodules ( < 0.001) . Specifically, treatment of osteoblastlike cells with anti-IGF I or anti-IGF II resulted in 7.0-fold and 5.4-fold declines in mean bone nodule formation compared to cells without such treatment . CONCLUSIONS: Intramedullary reaming prior to pin insertion resulted in a significantly greater number of bone nodules than pin insertion only . Antibodies to IGF I, IGF II, and indomethacin reversed the stimulatory effect of reaming on bone nodule formation, suggesting their role in modulating the course of fracture healing following intramedullary reaming.

Biotechnol Bioeng, 2003 Jan 5, 81(1), 33 - 49
Metabolic flux analysis of cultured hepatocytes exposed to plasma; Chan C et al.; Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution . The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices . During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions . In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure . Furthermore, supplementing the plasma with amino acids restored hepatospecific functions . In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation . We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis . These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes . The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma . Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma . The increase in fatty acid oxidation reduced intracellular triglyceride accumulation . Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture .

Neurosci Lett, 2002 Dec 6, 334(1), 33 - 6
Quantification of axotomized ganglion cell death by explant culture of the rat retina; Manabe S et al.; We first demonstrated a temporal profile of retinal ganglion cell (RGC) death after axotomy in situ using a newly developed retinal explant culture system . 1,1'- dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent tracer, was administered to the superior colliculi of 2 day old Wistar rats to label RGCs retrogradely . Small pieces of retinas were dissected and maintained at the interface between a 5% CO(2) atmosphere and culture media, and temporally observed by fluorescent microscopy . The number of surviving RGCs, identified as fluorescent spots, gradually decreased during the course of experiments for up to 10 days in vitro . We identified apoptotic RGCs by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling . Administration of cycloheximide, actinomycin D, or a caspase-3 inhibitor to media significantly decreased RGC death . This system provides a method of quantifying axotomized RGC death in relation to time-dependent changes in an identical retinal slip .

Int J Oncol, 2002 Dec, 21(6), 1259 - 67
Mifepristone-induced secretion of transforming growth factor beta1-induced apoptosis in prostate cancer cells; Liang Y et al.; Successful therapy should induce apoptosis in prostate cancer cells irrespective of their androgen response . We have investigated the possibility of utilizing Mifepristone and Tamoxifen as treatment options for prostate cancer cells . Because preliminary results demonstrated induction of apoptosis by these drugs, the mechanism of induction of apoptosis was investigated . LNCaP-C4 prostate cancer cells were treated with Mifepristone and/or Tamoxifen . To confirm cytotoxic effects, nude mice with LNCaP-C4 xenografts were treated with Mifepristone and Tamoxifen . Cell viability was assayed using Sulforhodamine B (SRB) assay and DNA fragmentation was measured by ELISA . Culture media from vehicle- and drug-treated cells were collected and secretion of transforming growth factor beta1 (TGFbeta1) was estimated by ELISA . Role of TGFbeta1 was confirmed by inhibiting its function using TGFbeta1 antibody or M6P, which blocked activation of TGFbeta1 . Apoptotic effects were determined by immunoblots of cytochrome c levels in cytosol and by in vitro colorimetric assay of caspase-3 activity . Results showed that although both drugs induced apoptosis in LNCaP-C4 cells, Mifepristone was more effective . The effects of these drugs on xenografts confirmed in vitro results . It was hypothesized that drug-induced secretion of TGFbeta1 may be responsible for induction of apoptosis . Neutralization of TGFbeta1 with an antibody or blocking the activation of TGFbeta1 by M6P abrogated the effects of Mifepristone and Tamoxifen confirming our hypothesis . Furthermore, treatment with Mifepristone and/or Tamoxifen released cytochrome c into the cytoplasm and induced activity of caspase-3, providing evidence that the drug-stimulated secretion of TGFbeta1 was responsible for induction of apoptosis in these cells . In conclusion, both Mifepristone and Tamoxifen induced apoptosis mediated through TGFbeta1 . However, no critical advantage was noted by the addition of Tamoxifen to Mifepristone treatment.

Environ Health Perspect, 2002 Oct, 110 Suppl 5, 761 - 6
Sodium arsenite-induced stress-related gene expression in normal human epidermal, HaCaT, and HEL30 keratinocytes; Trouba KJ et al.; Arsenic is a carcinogen that poses a significant health risk in humans . Based on evidence that arsenic has differential effects on human, rodent, normal, and transformed cells, these studies addressed the relative merits of using normal human epidermal keratinocytes (NHEK) and immortalized human (HaCaT) and mouse (HEL30) keratinocytes when examining stress-induced gene expression that may contribute to carcinogenesis . We hypothesize that redox-related gene expression is differentially modulated by arsenic in normal versus immortalized keratinocytes . To test the hypothesis, we exposed keratinocytes to sodium arsenite for 4 or 24 hr, at which time serine threonine kinase-25 (stk25) and nicotine adenine dinucleotide phosphate {nad(p)h} quinone oxidoreductase gene expression were measured . The effect of glutathione reduction on arsenite-induced cytotoxicity and gene expression in NHEK also was evaluated by addition of l-buthionine-{S,R}-sulfoximine (BSO) to culture media . Results indicate the term LC(50) for arsenite is approximately 10-15 microM in NHEK and HEL30 keratinocytes and 30 microM in HaCaT keratinocytes . Compared with HaCaT and HEL30 keratinocytes, a nontoxic concentration of arsenite (2.5 microM) increases stk25 and nad(p)h quinone oxidoreductase gene expression in NHEK, an effect partially attenuated by BSO . These data indicate that NHEK and HaCaT/HEL30 keratinocytes have similar sensitivities toward arsenite-induced cytotoxicity but unique gene expression responses . They also suggest that arsenite modulates gene expression in NHEK involved in cellular signaling and other aspects of intermediary metabolism that may contribute to the carcinogenic process.

Zhonghua Yi Xue Za Zhi, 2002 Sep 25, 82(18), 1232 - 4
{Effect of mitogen-activated protein kinase signal transduction in non-estrogen antagonistic mechanism of tamoxifen}; Yang D et al.; OBJECTIVE: To investigate the effect of mitogen-activated protein kinase (MAPK) signal cascade in the non-estrogen antagonistic mechanism of tamoxifen (TAM) . METHODS: Human breast cancer cells MCF-7 were cultured . TAM, PD98075, inhibitor of MAPK kinase (MEK), or TAM + PD98075 was added into the culture media, followed by methyl thiazolyl tetrazolium (MTT) and DMSO . Then the 542nm absorption value was measured and the growth curve was drawn . Western blot was used to measure the expression of p-extracellular signal-regulated kinase (ERK) in MCF-7 cells . Flow cytometry was applied to analyze the cell cycle and apoptosis . RESULTS: The optical density representing the relative expression of p-ERK was lower successively in the control, TAM, PD09875, and TAM + PD09875 groups . The apoptotic rate of MCF-7 cells was 6.44%, 8.3%, 36.5% and 53.5% in the control, PD98075, TAM, and TAM + PG98075 groups respectively The rate of cells in G(0)G(1) phase was 74.25%, 79.76%, 84.02%, and 95.82% in those groups respectively . Ther rate o cells in S phase was 21.03%, 15.22%, 11.43%, and 2.22% respectively in those groups . The rate of cells in G(2)M phase was 4.71%, 5.02%, 4.52%, and 1.96% respectively in those groups . CONCLUSION: MAPK signal transduction pathway plays a certain role in the non-estrogen antagonistic mechanism of tamoxifen.

Mol Reprod Dev, 2003 Jan, 64(1), 70 - 8
Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan; Lane M et al.; Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage . Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media . Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion . Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation . The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation . Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions . For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable .

J Vet Med Sci, 2002 Oct, 64(10), 887 - 91
Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo; Imai K et al.; The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes . No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells . Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05) . The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199 . Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05) . These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells . But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.

Reproduction, 2002 Nov, 124(5), 633 - 41
Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH; Buzzard JJ et al.; Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal . This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth) . Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface . The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5% . Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry . Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media . After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence . The method described provides a useful tool for investigating the control of Sertoli cell division . Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.

J Rheumatol, 2002 Nov, 29(11), 2261 - 5
Increased expression of arginase II in patients with different forms of arthritis . Implications of the regulation of nitric oxide; Corraliza I et al.; OBJECTIVE: To investigate the expression of arginase isoforms in patients with different forms of arthritis and the possible implications of the synthesis of nitric oxide (NO) . METHODS: Arginase activity was measured in synovial fluid (SF) cells from patients with different forms of arthritis, either directly or after in vitro stimulation with cytokines . The identity of the isoform expressed was confirmed by reverse transcription polymerase chain reaction . We measured both arginase activity and NO production in SF macrophages and synovial membrane fibroblasts from patients with rheumatoid arthritis (RA) . RESULTS: Arginase II was the isoform expressed in SF cells . In SF macrophages, dibutyryl-cAMP (dBt-cAMP), prostaglandin E2 (PGE2), and lipopolysaccharide (LPS) further increased the enzyme activity, while NO production was not detected even in the presence of Th1-like cytokines . In contrast, synovial membrane fibroblasts from patients with RA released NO into the culture media . Moreover, dBt-cAMP, PGE2, and transforming growth factor-beta, which induced arginase II, reduced the levels of NO . Reciprocally, the induction of NO by Th1 cytokines inhibited arginase activity levels . CONCLUSION: Arginase II expression is upregulated in RA and may increase cell proliferation by providing L-ornithine, which is the substrate of polyamine biosynthesis . In cells where both arginase II and inducible NO synthase activity occurs, there is a reciprocal regulation, suggesting that agents that induce arginase II in synovial cells could downregulate the levels of NO and divert L-arginine metabolism toward cell proliferation and/or tissue regeneration.

J Clin Endocrinol Metab, 2002 Nov, 87(11), 5209 - 19
Cyclic mechanical stretch augments prostacyclin production in cultured human uterine myometrial cells from pregnant women: possible involvement of up-regulation of prostacyclin synthase expression; Korita D et al.; Prostacyclin (PGI(2)), a potent smooth muscle relaxant, is a major prostaglandin secreted from human myometrium . The concentrations of PGI(2) metabolites in the maternal plasma were reported to be elevated during pregnancy, especially in labor . To clarify the mechanism in PGI(2) secretion from the myometrium, we first investigated the protein expression of cytosolic phospholipase A(2), cyclooxygenase (COX)-1, COX-2, and prostacyclin synthase (PGIS) in the human uterine myometrium at various gestational ages before labor . To elucidate the involvement of labor in the increase in PGI(2) production during labor, we next examined the effect of labor-like cyclic mechanical stretch on PGI(2) production by cultured human myometrial cells . Pregnancy specifically increased COX-1 and PGIS protein expression in the myometrial tissues before labor (P < 0.01 for both) . Cyclic mechanical stretch augmented PGIS promoter activity, via activation of activator protein-1 site, and PGIS mRNA and protein expression in cultured human myometrial cells and resulted in a 3.5-fold increase in the concentration of 6-keto-prostaglandin F(1alpha), the stable metabolite of PGI(2), in the culture medium (P < 0.05) . However, stretch did not affect the levels of prostaglandin E(2), prostaglandin F(2alpha), or thromboxane A(2) secreted into the same culture media . These results suggest that cyclic mechanical stretch during labor may contribute to the increase in the PGI(2) concentration in the maternal plasma during parturition.

Clin Exp Rheumatol, 2002 Sep-Oct, 20(5), 669 - 76
Influence of polysulphated polysaccharides and hydrocortisone on the extracellular matrix metabolism of human articular chondrocytes in vitro; Wang L et al.; OBJECTIVE: To evaluate the influence of hydrocortisone and two polysulphated polysaccharides (xylosan polysulphate and chondroitin polysulphate) on the extracellular matrix metabolism of chondrocytes cultured in gelled agarose . METHODS: Isolated chondrocytes from normal femoral cartilage of the knee joints of 7 donors were cultured in gelled agarose to maintain their differentiated phenotype . After two weeks of culture, hydrocortisone (0.2 microgram/ml), xylosan polysulphate (10 micrograms/ml) and chondroitin polysulphate (10 micrograms/ml) were added to the culture media supplemented with or without interleukin (IL)-1 beta . After one week of incubation, the cells were liberated from the agarose with agarase . Isolated cells were labelled with antibodies against aggrecan and type II collagen, as well as biotinylated hyaluronic acid binding protein to analyse the extracellular matrix (ECM) molecules in the cell-associated matrix (CAM) . The levels of matrix metalloproteinase (MMP)-1, -3, and -13, as well as tissue inhibitor of metalloproteinase (TIMP)-1 and -3 were determined after the cells had been permeabilised and stained with the appropriate antibodies . Triplicate samples were analysed with flow cytometry . RESULTS: IL-1 beta decreased the accumulation of aggrecan, hyaluronan and type II collagen in the CAM and increased intracellular MMP-1, -3 and -13 at a concentration of 100 pg/ml . Xylosan polysulphate and chrondroitin polysulphate restored the expression of these CAM molecules in these IL-1 beta-treated cultures . Hydrocortisone stimulated the accumulation of CAM aggrecan and hyaluronan whether or not under the exposure to IL-1 beta . Intracellular MMP-1, -3, -13 and TIMP-1 and -3 of IL-1 beta-treated cells was downregulated after treatment with hydrocortisone . CONCLUSION: Both hydrocortisone and the two polysulphated polysaccharides could stimulate the accumulation of CAM macromolecules of IL-1 beta-treated chondrocytes . This effect probably resulted in part from the downregulation of MMPs . These agents showed cartilage structure modifying effects in vitro.

Br J Pharmacol, 2002 Nov, 137(6), 901 - 9
Inhibitory mechanism of vascular endothelial growth factor (VEGF) by bucillamine; Koyama S et al.; 1 . Vascular endothelial growth factor (VEGF) plays an important role in the neovascularization of ischaemic retinal diseases such as proliferative diabetic retinopathy . We determined that bucillamine, an anti-rheumatic drug, inhibits the VEGF production induced by hypoxia in bovine retinal microcapillary endothelial cells (BREC) . To further clarify the inhibitory mechanism, we investigated the possible mechanism by which bucillamine exerts this inhibitory effect . 2 . Bucillamine (100 micro M) decreased the hypoxia-induced increase of VEGF mRNA by 54.5% (P<0.001) . Bucillamine (100 micro M) reduced the hypoxia-induced VEGF content in culture media by 29.0% (P<0.001), while monosulfydryl drugs, N-acetylcysteine and D-penicillamine, did not . 3 . Bucillamine (100 micro M) did not affect VEGF mRNA half-life (hypoxia, 4.3 h; hypoxia+bucillamine, 3.9 h; normoxia, 2.7 h; normoxia+bucillamine, 2.7 h) . 4 . Reporter gene studies revealed that bucillamine reduced transcriptional activity in the 5'-flanking region of the VEGF gene by 74.0% . Hypoxia stimulated binding activity of BREC nuclear protein to a hypoxia responsive element (HRE), which was decreased by bucillamine . 5 . Bucillamine inhibited hypoxic-induction of HIF-1alpha mRNA by 73.1% (P<0.001) . Bucillamine also inhibited spontaneous VEGF mRNA expression by 26.6% . Furthermore, it inhibited activity of VEGF promoter and decreased binding activity to Sp1 and HRE, but did not alter AP1 and AP2 activity in normoxia . 6 . These data suggest that bucillamine inhibits hypoxic induction of VEGF through inhibition of HIF-1 induction and binding activity in BREC . Bucillamine also inhibits the spontaneous expression of VEGF mRNA by its effect on Sp1 and HRE binding.

Eur J Neurosci, 2002 Oct, 16(7), 1275 - 83
Astrocytes enhance lipopolysaccharide-induced nitric oxide production by microglial cells; Sola C et al.; Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells . The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment . However, NO production in endotoxin-treated astrocyte cultures is controversial . We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media . We also identified the NO-producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells . Only microglial cells showed iNOS immunoreactivity . Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures . We then analysed the effect of astrocytes on NO production by microglial cells using microglial-astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells . Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS-treated astrocytes could account, at least in part, for this enhancement.

Reprod Toxicol, 2002 Nov-Dec, 16(6), 795 - 800
Effects of nicotine and cotinine on bovine theca interna and granulosa cells; Sanders SR et al.; The purpose of this study was to determine if nicotine or cotinine inhibits steroidogenesis in the ovarian follicle . Theca interna and granulosa cells were isolated from bovine follicles, cultured with nicotine or cotinine for 24h, and culture media were assayed for androstenedione or estradiol . Treatment of theca interna with 6, 60, and 600 micro M nicotine decreased (P<or=0.002) production of androstenedione to 55, 53, and 24% of control levels, respectively . Levels of androstenedione in theca interna treated with cotinine were not different from control values . In granulosa cells, nicotine inhibited production of estradiol at the highest dose tested . Treatment with 600 micro M nicotine decreased (P<or=0.001) estradiol concentration to 12% of control values, attributable to a general cytotoxic effect . Cotinine had no effect on estradiol production by granulosa cells . These results provide novel evidence for inhibitory effects of nicotine on androgen production by theca interna.

Mol Hum Reprod, 2002 Nov, 8(11), 992 - 7
Regulation of follistatin-related gene (FLRG) expression by protein kinase C and prostaglandin E(2) in cultured granulosa-luteal cells; Liu J et al.; Activin and its binding protein follistatin may act as local regulators of cell growth and steroidogenesis in the human ovary . The recently identified follistatin-related gene (FLRG) is expressed abundantly in the human ovary, has high affinity for activin, and is able to inhibit activin-induced transcriptional responses . However, little is known about the regulation of FLRG expression in specific cell types in the ovary, while it is known that gonadotrophins induce follistatin gene expression in human granulosa-luteal cells . In this study, we investigated the expression of FLRG mRNA in granulosa-luteal cells of preovulatory follicles obtained from women undergoing IVF . FLRG mRNA was detected by RT-PCR in fresh and cultured granulosa-luteal cells, as well as in normal ovarian stroma, theca and granulosa cells . Northern blot analysis revealed a 2.5 kb transcript of the FLRG in cultured granulosa-luteal cells . The protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 160 nmol/l), and prostaglandin E(2) (PGE(2), 1 micromol/l) increased FLRG mRNA accumulation up to 3-8 fold over the control level after 24 h of treatment, and these stimulatory effects were dose-dependent . Co-treatment with the protein kinase C inhibitor, Ro-31-8220 (3 micromol/l), blocked the stimulatory effect of TPA . Although short term treatment with the protein kinase A activator, (Bu)(2)cAMP (1 mmol/l), slightly reduced FLRG mRNA expression in most experiments, long term treatment with FSH (100 IU/l), LH (100 IU/l), or (Bu)(2)cAMP had no significant effect on the FLRG mRNA levels . As expected, gonadotrophins, protein kinase A and C activators and PGE(2) increased granulosa-luteal cell progesterone secretion into the culture media . Taken together, previous and our present data suggest that protein kinase C and A signal transduction pathways differently regulate the expression of FLRG and follistatin genes in human ovarian granulosa-luteal cells.

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