Fetal Calf Serum Medium Research Articles

Metabolic Profiling of SH-SY5Y and Neuro2A Cells in Relation to Fetal Calf Serum (FCS) Concentration in Culture Media.

The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5-10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments.

Genetic profiling of human bone marrow mesenchymal stromal cells after in vitro expansion in clinical grade human platelet lysate.

Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded ex vivo. In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in vitro in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells in vitro. Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs). RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.

In Vitro Effects of Natrum muriaticum in Kidney Cell Lines MDCK and LLC-PK1

Previous papers have indicated that homeopathic solutions modify the cellular and biochemical aspects of cells maintained in culture. In this study, the effects of Natrum muriaticum, a medicine used in the homeopathic clinic for the treatment of hypertension, were evaluated in kidney MDCK and LLC-PK1 cell lines. The following cellular parameters were analyzed: viability, morphology and expression of the (Na+-K+)-ATPase and the angiotensin II receptors AT1 and AT2. The cell lines were plated (5.0 x 104 cells/mL) in DMEM supplemented with 10% fetal calf serum (FCS). After 24 hours at 37°C, DMEM was re-fed with the addition of 10% (V/V) and 1% (V/V) of the following samples: Natrum muriaticum 30CH, water 30CH and non-dynamized sterile distilled water to do the MTT assay. The results obtained from these groups were compared to those obtained by incubation of the cells in culture medium free of these solutions (Control). Cell viability was assessed by a colorimetric MTT ELISA assay (490nm). The values from four independent experiments performed in quintuplicate were plotted and statistically analyzed by Sigma Plot v.11 (Jandel Scientific). The morphology of MDCK cells was evaluated by optical microscope after Giemsa’s staining. The expression of the (Na++K+)-ATPase and AT1/AT2 of LLC-PK1 cells was evaluated by Western Blot (WB) analysis. For this experimental set, 5.0x104 cells/mL were incubated in DMEM supplemented with 10% FCS and daily culture medium was replaced by a new one, containing: Natrum muriaticum 30CH and water 30CH. Additionally, cells were treated for 5, 10 and 15 days with 1% of specific solutions and the total protein was measured by the Lowry method, after cell lysis. The samples were analyzed by electrophoresis in SDS-PAGE (12% gel) and transferred to nitrocellulose membrane. This membrane was incubated with specific primary antibodies (anti-(Na++K+)-ATPase, anti-AT1 and AT2 or anti-anti-beta-actin). The detection was performed using ECL system and Hyperfilm. MTT assays showed a statistically significant reduction in cellular mitochondrial activity (p

Primary Human Fibroblasts in Culture Switch to a Myofibroblast-Like Phenotype Independently of TGF Beta.

Fibroblasts are the prevalent cell type and main source for extracellular matrix (ECM) in connective tissue. Depending on their origin, fibroblasts play a central role in non-pathological tissue remodeling and disease like fibrosis. This study examined the effect of established culture conditions of primary human fibroblasts, from different origins on the myofibroblast-like phenotype formation. We isolated primary human fibroblasts from aortic adventitia, lung, juvenile- and adult skin and investigated the expression levels of CD90, alpha smooth muscle actin (αSMA) and procollagen I under different concentrations of fetal calf serum (FCS) and ascorbic acid (AA) in culture media by immunoblot and immunofluorescence assays. Furthermore, we determined the viability using XTT and migration/wound healing in scratch assays. Collagen 1 secretion was quantified by specific ELISA. Primary human fibroblasts show in part a myofibroblast-like phenotype even without addition of FCS. Supplemented AA reduces migration of cultured fibroblasts with no or low concentrations of FCS. Furthermore, AA and higher concentrations of FCS in culture media lead to higher levels of collagen 1 secretion instead of procollagen I accumulation. This study provides evidence for a partial switch of primary human fibroblasts of different origin to a myofibroblast-like phenotype under common culture conditions.

Production of farnesol in Candida albicans biofilms of resistant and standard strains in different media

Objective To compare the production of farnesol between Candida albicans (C. albicans) biofilms formed by resistant and standard strains in different media, and to investigate the changing trend of farnesol production in different phases of biofilm formation and the features of farnesol production by resistant C. albicans. Methods Fluconazole-resistant C. albicans strains were induced in vitro. Standard strains and fluconazole-resistant strains of C. albicans were separately inoculated onto different media, including RPMI 1640 medium, yeast extract peptone dextrose (YPD) medium, yeast nitrogen base (YNB) + 0.5% glucose medium, RPMI 1640 + 10% fetal calf serum (FCS) , so as to form C. albicans biofilms. Morphological changes of C. albicans biofilms at 24 hours were observed under an inverted microscope, and gas chromatography-mass spectrometry (GC/MS) was performed to detect the level of farnesol at 1.5, 3, 6, 12, 24, 36 and 48 hours. Results There were no obvious differences in the morphology of C. albicans biofilms between the resistant and standard strains when they were cultured in the same medium, while the morphology of C. albicans biofilms markedly differed between the 2 kinds of strains in the different media. Three-factor analysis of variance showed that the production of farnesol in the C. albicans biofilms changed over time (F= 70.628, P < 0.001) . Concretely speaking, during the formation of resistant and standard C. albicans biofilms, the production of farnesol gradually increased in the RPMI 1640, YPD and YNB+ 0.5% glucose media until the biofilms matured, then showed a decreasing trend. However, the time to peak levels of farnesol was different between the 2 kinds of strains in these media. Moreover, the levels of farnesol in the 2 kinds of strains both slowly increased in the RPMI 1640+ 10% FCS medium within 12-48 hours. Culture media also significantly affected the production of farnesol (F= 176.665, P < 0.001) , and the levels of farnesol in the resistant and standard C. albicans biofilms were both higher in the YNB+ 0.5% glucose medium. When resistant and standard strains were separately cultured in the RPMI 1640 media and the YPD media, the level of farnesol was significantly higher in the resistant strains than in the standard stains (RPMI 1640 media at 36 hours: 1.157 ± 0.064 vs. 0.250 ± 0.075, P < 0.05; YPD media at 6 hours: 0.262 ± 0.036 vs. 0.055 ± 0.062, P < 0.05; YPD media at 12 hours: 0.730 ± 0.030 vs. 0.482 ± 0.024, P < 0.05) . However, when they were separately cultured in the YNB+ 0.5% glucose media, the farnesol level was significantly higher in the standard stains than in the resistant strains (36 hours: 2.950 ± 0.677 vs. 0.523 ± 0.266, P= 0.020) . Conclusion The media markedly affect the production of farnesol in the C. albicans biofilms, and there is a certain difference in the production of farnesol between resistant and standard C. albicans strains. Key words: Candida albicans; Biofilms; Drug resistance, fungal; Farnesol; Culture media; Gas chromatography-mass spectrometry

THE EFFECT OF FETAL CALF SERUM ON HUMAN DENTAL PULP STEM CELLS

Authors studied potential side effects of fetal calf serum (FCS) in cultivation media on human dental pulp stem cells (DPSC) during long-term cultivation. Two lines of DPSC obtained healthy donors (male 22 years, female 23 years) were used. Both lines were cultivated under standard cultivation conditions in four different media containing 10% or 2% FCS and substituted with growth factors. During long-term cultivation proliferation ability, karyotype and phenotype of DPSC were measured. Both lines of DPSC cultivated in a media containing 2% FCS and ITS supplement showed the highest number of population doublings. On the other hand the proliferation rate of DPSC cultivated in a media with 2% FCS without ITS supplement was slowest. Proliferation rate of DPSC cultivated in 10% FCS media with or without FGF-2 was comparable. DPSC cultivated in a media with 10% FCS showed a significantly higher amount of chromosomal aberrations. These chromosomal aberrations do not seem to be clonal but surprisingly we found large amounts of tetraploid cells in the 9th passage in both media containing 10% FCS. Our study proved that cultivation of DPSC in media containing higher concentration of FCS has critical side effects on cell chromosomal stability.

Effect of serum concentration on the cytotoxicity of clay particles

Nanoparticle cytotoxicity testing based on in vitro methods frequently lack consistency. Even the inclusion of the commonly employed growth supplement, FCS (fetal calf serum), generates variable results. Thus, our object was to investigate the effect of FCS concentration on the cytotoxic behaviour of the unmodified nanoclay, Cloisite® Na(+). Human monocytic U937 cells in medium supplemented with 5% FCS, 2.5% FCS or serum-free medium were treated with 1 mg/ml Cloisite Na(+). Cell growth in 2.5% FCS was significantly inhibited by Cloisite Na(+) within 48 h, whereas little effect was seen with a supplement of 5% FCS. Without serum, cell growth was inhibited and Cloisite Na(+) had a detrimental effect on these cells. In media supplemented with FCS, the nanoclays agglomerated together to form large bundles, whereas they were evenly dispersed throughout the medium in the absence of serum. Clay particles, therefore, have cytotoxic properties that may be linked to their dispersion pattern. These adverse effects seem to be masked by 5% FCS. Serum supplementation is an important consideration in the toxicological assessments of nanomaterials on cells, which needs to be addressed in the standardization of in vitro testing methods.

Translation Initiator EIF4G1 Mutations in Familial Parkinson Disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.

137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P &gt; 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P &lt; 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P &lt; 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P &gt; 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP.

Comparison of three culture media for the establishment of melanoma cell lines

Melanoma cell lines are useful tools for the analysis of tumor-specific lymphocytes which are injected to patients treated by adoptive immunotherapy. So they have been established previously (with an efficacy of 47%) in Roswell Park Memorial Institute (RPMI) medium enriched with fetal calf serum (FCS). In order to improve the probability of establishing melanoma cell lines, we compared two FCS-free media with the original FCS medium. Ten melanoma-invaded lymph nodes were tested for their ability to grow in three different culture media: RPMI with FCS; RPMI with human serum (HS); serum-free X-vivo 15 (X15). For each medium, we compared the following criteria: percentage of lines obtained; period of establishment; cell morphology; expression of melanoma-associated antigens and surface molecules. More cell lines were obtained with HS and X15 media compared to FCS medium (7/10, 5/10 and 4/10, respectively). The time period to establish a stable line was similar for the three media. No morphological differences were observed in cells derived from the same tumor sample in the different media. With the X15 medium, cells generally expressed lower levels of melanocytic differentiation antigens and surface molecules. The growth of melanoma cell lines in FCS-free culture media appears possible and advantageous, with an increased probability of obtaining autologous tumor cell lines. Furthermore the cells obtained could be used as multiple antigenic sources in active or adoptive immunotherapy protocols.

298 THE INFLUENCE OF DISTINCT CENTRAL BULL STATIONS ON THE IN VITRO EMBRYONIC DEVELOPMENT OF NELORE BULLS

Reproductive biotechnology is growing worldwide as one of the most important tools of cattle breeding because it accelerates the process of genetic improvement. Most of the embryos produced are obtained using frozen semen from different AI centers. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions that occur during cooling and ice formation. It has previously been described that bad management of frozen semen can result in reduced fertilization. This study investigated the influence of different central bull stations on the development of in vitro-produced bovine embryos. We compared semen of 154 Nelore bulls, used for IVF, from 8 different central bull stations (all of which used the same cryopreservation protocol) in the development of blastocysts. The in vitro production of embryos was performed as described: oocytes were collected from the slaughterhouse and matured in TCM-199 + 10% fetal calf serum (FCS) +0.5 μg mL-1 FSH + 50 μg mL-1 LH+ 1 μg mL-1 estradiol, for 24 hat 38.5°C in 5%CO2 in atmospheric air. Viable spermatozoids were obtained by centrifugation in Percoll gradient (45 and 90%), and used for IVF in a concentration of 2 million spermatozoa per mL in TALP + 10 μg mL-1 of heparin medium. After 12 h, the presumptive zygotes were transferred to a CR2+ 10% FCS medium and co-cultured with cumulus cells. After 168 h of IVF, we evaluated the number and stage of cleaved embryos produced with the semen of each bull. Statistical analyses were performed by using the chi-square test. Our results suggest that there are differences among distinct central bull stations in the proportion of embryos that developed into blastocysts and the different stages they hatched. FAPESP, CNPq, PROEX, FAEPA.

Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 degrees C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.

Can Platelet-Rich Plasma Enhance Tendon Repair?

Background Autologous platelet-rich plasma (PRP) application appears to improve tendon healing in traumatic tendon injuries, but basic knowledge of how PRP promotes tendon repair is needed. Hypothesis Platelet-rich plasma has a positive effect on cell proliferation and collagen production and induces the production of matrix-degrading enzymes and endogenous growth factors by human tenocytes. Study Design Controlled laboratory study. Methods Human tenocytes were cultured 14 days in 2% fetal calf serum medium complemented with 0%, 10%, or 20% vol/vol platelet-rich clot releasate ([PRCR] the active releasate of PRP) or platelet-poor clot releasate (PPCR). At day 4,7, and 14, cell amount, total collagen, and gene expression of collagen Iα1 (COL1) and Mod (COL3), matrix metalloproteinases ([MMPs] MMP1, MMP3, and MMP13), vascular endothelial-derived growth factor (VEGF)-A, and transforming growth factor (TGF)-β1 were analyzed. Results Platelet numbers in PRP increased to 2.55 times baseline. Growth-factor concentrations of VEGF and platelet-derived growth factor (PDGF)-BB were higher in PRCR than PPCR. Both PRCR and PPCR increased cell number and total collagen, whereas they decreased gene expression of COL1 and COL3 without affecting the COL3/COL1 ratio. PRCR, but not PPCR, showed upregulation of MMP1 and MMP3 expression. Matrix metalloproteinase 13 expression was not altered by either treatment. PRCR increased VEGF-A expression at all time points and TGF-β1 expression at day 4. Conclusion In human tenocyte cultures, PRCR, but also PPCR, stimulates cell proliferation and total collagen production. PRCR, but not PPCR, slightly increases the expression of matrix-degrading enzymes and endogenous growth factors. Clinical Relevance In vivo use of PRP, but also of PPP to a certain extent, in tendon injuries might accelerate the catabolic demarcation of traumatically injured tendon matrices and promote angiogenesis and formation of a fibrovascular callus. Whether this will also be beneficial for degenerative tendinopathies remains to be elucidated.

Generation of Immunosuppressive Mesenchymal Stem Cells in Allogeneic Human Serum

Mesenchymal stem cells (MSC) may be used to treat acute graft-versus-host disease and for tissue repair. In vitro expansion of MSC has been achieved in the presence of fetal calf serum (FCS). For safety and regulatory reasons, we explored if FCS could be replaced by human blood group AB serum. Proliferation and fold increase of MSC was higher in the presence of AB-serum, compared to FCS. Similar to cells generated in FCS media, MSC from AB-serum media were more than 95% positive for CD90, CD105 and human leukocyte antigen (HLA) class I, and negative for hematopoietic and endothelial markers CD14, CD31, CD34, CD45, and CD80. HLA class II expression was higher in MSC generated in AB-serum, but decreased with higher passage numbers. MSC generated in AB-serum suppressed lymphocyte proliferation in mixed lymphocyte cultures and after stimulation with phytohemagglutinin. MSC expanded in AB-serum and FCS have similar in vitro properties.

Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.

Ginsenosides promote meiotic maturation of mouse oocytes in cumulus-oocyte complexes involving increased expression of nitric oxide synthase

Ginseng has long been used in Asia because of its beneficial effects on human health. Ginsenosides are the primary biologically active components in ginseng. The aim of the present study was to determine the role of ginsenosides on meiotic maturation of murine oocytes to better understand the effects of ginsenosides on reproductive health. Cumulus-oocyte complexes (COCs) and denuded oocytes in germinal vesicle stage were isolated from equine chorionic gonadotropin-primed immature mice and were cultured for 24 hours in 10% fetal calf serum medium, which was supplemented with 4 mmol hypoxanthine to maintain meiotic arrest. Ginsenosides (1.0-10 μg/mL) overcame the hypoxanthine-maintained meiotic arrest. In addition, ginsenosides-stimulated meiotic maturation in COCs was assessed by increased germinal vesicle breakdown and first polar body extrusion. No effect of ginsenosides was observed on denuded oocytes. In addition, ginsenosides (1.0 μg/mL) could synergize with follicle-stimulating hormone (0.1 IU/mL) to promote oocyte meiotic maturation in COCs. Tamoxifen (an estrogen receptor α antagonist) did not influence the ginsenosides-induced meiotic maturation, suggesting the effect was not mediated by estrogenic action. However, a nitric oxide (NO) synthase inhibitor, N ω -nitro- l-arginine methyl ester ( l-NAME, 1 mmol), reduced the ginsenosides-induced oocyte maturation in COCs. Immunocytochemical analysis demonstrated that ginsenosides enhanced expression of inducible nitric oxide synthase (iNOS) in the cumulus cells. l-NAME reduced the ginsenoside-induced increase in iNOS immunoreaction. Our results indicate that ginsenosides might stimulate meiotic maturation of mouse oocytes through a paracrine pathway involving the NO/iNOS system.

Identification of an Hexapeptide That Binds to a Surface Pocket in Cyclin A and Inhibits the Catalytic Activity of the Complex Cyclin-dependent Kinase 2-Cyclin A

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Proline-rich Motifs in the Parathyroid Hormone (PTH)/PTH-related Protein Receptor C Terminus Mediate Scaffolding of c-Src with β-Arrestin2 for ERK1/2 Activation

Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and beta-arrestin2-mediated internalization. Beta-arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G(q)-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with beta-arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative beta-arrestin2. Third, the c-Src inhibitor PP2 and a kinase-dead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and beta-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with beta-arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and beta-arrestin2 with a class II G-protein-coupled receptor.

High Yields of Autologous Living Dermal Equivalents Using Porcine Gelatin Microbeads as Microcarriers for Autologous Fibroblasts

Permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. This study was to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous living dermal equivalents on a large scale. Autologous fibroblasts were isolated from split-thickness skin biopsy from a leg ulcer patient, inoculated onto macroporous porcine gelatin microbeads, and incubated in a bioreactor (Cellspin) in serum-free fibroblast growth medium or in DMEM medium containing 10% fetal calf serum (FCS). Fibroblasts rapidly adhered to and actively proliferated on the microbeads in the bioreactor in both serum-free and serum-containing medium. MTT assay showed the number of fibroblasts on the microbeads reached up to 5.3- or 4.0-fold the cells seeded in DMEM medium containing 10% FCS or serum-free medium, respectively. When removed from Bio-MCCS and cultured under static conditions, fibroblasts were able to leave the microbeads and proliferate to confluence on the bottom of tissue culture flasks. When stored at room temperature in DMEM containing 10% FBS, fibroblast cultured on the microbeads retained highest viabilities for at least 3 weeks, up to 82% of originals. This Bio-MCCS using porcine gelatin microbeads as carriers for fibroblasts offers a new option of mass production of autologous living dermal equivalents.

Essential Role of Synoviolin in Embryogenesis

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.