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Sample records for bovine cumulus cells

  1. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes

    NARCIS (Netherlands)

    Ortiz-Escribano, N.; Smits, K.; Piepers, S.; Abbeel, Van den E.; Woelders, H.; Soom, Van A.

    2016-01-01

    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were

  2. Bovine cumulus-granulosa cells contain biologically active retinoid receptors that can respond to retinoic acid

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    Malayer Jerry

    2003-11-01

    Full Text Available Abstract Retinoids, a class of compounds that include retinol and its metabolite, retinoic acid, are absolutely essential for ovarian steroid production, oocyte maturation, and early embryogenesis. Previous studies have detected high concentrations of retinol in bovine large follicles. Further, administration of retinol in vivo and supplementation of retinoic acid during in vitro maturation results in enhanced embryonic development. In the present study, we hypothesized that retinoids administered either in vivo previously or in vitro can exert receptor-mediated effects in cumulus-granulosa cells. Total RNA extracted from in vitro cultured cumulus-granulosa cells was subjected to reverse transcription polymerase chain reaction (RT-PCR and mRNA expression for retinol binding protein (RBP, retinoic acid receptor alpha (RARalpha, retinoic acid receptor beta (RARbeta, retinoic acid receptor gamma (RARgamma, retinoid X receptor alpha (RXRalpha, retinoid X receptor beta (RXRbeta, retinaldehyde dehydrogenase-2 (RALDH-2, and peroxisome proliferator activated receptor gamma (PPARgamma. Transcripts were detected for RBP, RARalpha, RARgamma, RXRalpha, RXRbeta, RALDH-2, and PPARgamma. Expression of RARbeta was not detected in cumulus-granulosa cells. Using western blotting, immunoreactive RARalpha, and RXRbeta protein was also detected in bovine cumulus-granulosa cells. The biological activity of these endogenous retinoid receptors was tested using a transient reporter assay using the pAAV-MCS-betaRARE-Luc vector. Addition of 0.5 and 1 micro molar all-trans retinoic acid significantly (P trans retinol stimulated a mild increase in reporter activity, however, the increase was not statistically significant. Based on these results we conclude that cumulus cells contain endogenously active retinoid receptors and may also be competent to synthesize retinoic acid using the precursor, retinol. These results also indirectly provide evidence that retinoids

  3. Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production.

    Science.gov (United States)

    Rispoli, L A; Payton, R R; Gondro, C; Saxton, A M; Nagle, K A; Jenkins, B W; Schrick, F N; Edwards, J L

    2013-08-01

    When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

  4. Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells

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    Looft C

    2011-01-01

    Full Text Available Abstract Background The bi-directional communication between the oocyte and its companion cumulus cells (CCs is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. Results We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO + CCs and without (OO - CCs CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs + OO or without (CCs - OO their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs + OO and CCs - OO, respectively. While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18, translation (EIF2AK1, EIF4ENIF1 and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI, protein metabolic processes (IHH, APOA1, PLOD1, steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7. Similarly, while transcripts over expressed in OO + CCs

  5. The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

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    Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

    2017-10-01

    The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

  6. In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.

    2017-01-01

    In vitro production (IW) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level...

  7. Remodeling of Donor Nuclei, DNA-Synthesis, and Ploidy of Bovine Cumulus Cell Nuclear Transfer Embryos: Effect of Activation Protocol

    Czech Academy of Sciences Publication Activity Database

    Alberio, R.; Brero, A.; Motlík, Jan; Cremer, T.; Wolf, E.; Zakhartchenko, V.

    2001-01-01

    Roč. 59, č. 2 (2001), s. 371-379 ISSN 1040-452X R&D Projects: GA AV ČR KSK5052113 Grant - others:WO(DE) 685/2-1; WO(DE) 685/3-1 Keywords : nuclear transfer * cumulus cells * activation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.296, year: 2001

  8. Cumulus cell transcripts transit to the bovine oocyte in preparation for maturation

    DEFF Research Database (Denmark)

    Macaulay, Angus D.; Gilbert, Isabelle; Scantland, Sara

    2016-01-01

    So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and...

  9. Bovine cumulus-oocyte disconnection in vitro

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul

    1987-01-01

    Cumulus-oocyte complexes were obtained from cows by aspiration of small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and processed for transmission electron microscopy after different periods of culture including a 0...

  10. Enhancement of Bovine oocyte maturation by leptin is accompanied by an upregulation in mRNA expression of leptin receptor isoforms in cumulus cells

    NARCIS (Netherlands)

    van Tol, Helena T A; van Eerdenburg, Frank J C M; Colenbrander, Ben; Roelen, Bernard A J

    In this study, the mechanisms of supposed leptin action on oocyte maturation were examined. Expression of leptin mRNA, as determined with RT-PCR, was present in oocytes but not in cumulus cells. The long isoform of the leptin receptor (ObR-L) was expressed exclusively in cumulus cells after 7 and 23

  11. Interação entre células do cumulus e atividade da proteína quinase C em diferentes fases da maturação nuclear de oócitos bovinos Interaction between cumulus cells and the activity of protein kinase C at different stages of bovine oocyte nuclear maturation

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    A.C. Bertagnolli

    2004-08-01

    Full Text Available Verificou-se a influência da proteína quinase C (PK-C no reinício e na progressão da meiose em oócitos bovinos, determinando se as células do cumulus são mediadoras da PK-C na regulação da maturação dos oócitos. Complexos cumulus-oócitos (CCO e oócitos desnudos (OD, distribuídos aleatoriamente em seis tratamentos (T com base na presença de um ativador da PK-C (PMA (T1 e T2, de um forbol éster incapaz de ativar a PK-C (4alfa-PDD-controle (T3 e T4 ou de apenas o meio básico (TCM-199-controle (T5 e T6, foram cultivados por 7, 9, 12, 18 e 22 horas. A percentagem de rompimento da vesícula germinativa no grupo cultivado com PMA foi maior do que nos dois grupos controle, com e sem células do cumulus. O cultivo de CCO e OD por 12 e 18 horas demonstrou que a PK-C influencia a progressão para os estádios de metáfase I (MI e metáfase II (MII de maneira dependente das células do cumulus. Nos períodos de 9 e 22 horas, não foi possível observar diferença entre os grupos quanto aos diferentes estádios de maturação. A ativação da PK-C acelera o reinício da meiose independentemente das células somáticas e acelera a progressão até os estádios de MI e MII na dependência das células do cumulus.The aim of this study was to evaluate the effect of protein kinase C (PK-C on the meiotic resumption and progression in bovine oocyte, and to determine if the cumulus cells mediate the PK-C action in the regulation of bovine oocyte nuclear maturation. Cumulus-oocyte complexes (COC and denuded oocytes (DO, randomly allotted to 6 treatments (T based on the presence of an activator of PK-C (PMA (T1 and T2, or a phorbol ester unable to activate PK-C (4alphaPDD-control (T3 and T4 or a basic culture medium (T5 and T6, were cultivated for 7, 9, 12, 18 and 22 hours. The percentage of germinal vesicle breakdown (GVBD was higher when the oocytes were cultured with PMA than in the control groups with and without cumulus cells. However, PK-C was

  12. Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes in Vitro

    NARCIS (Netherlands)

    Pomar, F.J.; Roelen, B.A.J.; Slot, K.A.; Tol, van H.T.A.; Colenbrander, B.; Teerds, K.J.

    2004-01-01

    Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling

  13. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

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    Cheng-Jie Zhou

    2016-03-01

    Full Text Available Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes, in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes, and in vitro-matured, denuded oocytes without cumulus cells (DOs. Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

  14. Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex.

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    Héctor Alejandro Guidobaldi

    Full Text Available Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF, oviduct fluid, and conditioned medium from the cumulus oophorus (CU and the oocyte (O. Though several substances were confirmed as sperm chemoattractant, Progesterone (P seems to be the best chemoattractant candidate, because: 1 spermatozoa express a cell surface P receptor, 2 capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3 the CU cells produce and secrete P after ovulation; 4 a gradient of P may be kept stable along the CU; and 5 the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.

  15. Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

    Czech Academy of Sciences Publication Activity Database

    Ježová, M.; Scsuková, S.; Nagyová, Eva; Vranová, J.; Procházka, Radek; Kolena, J.

    2001-01-01

    Roč. 65, - (2001), s. 115-126 ISSN 0378-4320 R&D Projects: GA ČR GA524/98/0231; GA AV ČR KSK5052113 Keywords : pig-ovary * cumulus expansion * luteinization stimulator Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.196, year: 2001

  16. Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

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    Nevoral J.

    2015-01-01

    Full Text Available Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA, spectrophotometry, and high-performance liquid chromatography (HPLC in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

  17. Effect of Cumulus cell co-culture and Protein Supplement on Success of in vitro Fertilization and Development of Pre-implanted Embryos in mice

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    Muhammad-Baqir M-R. Fakhrildin

    2005-06-01

    Full Text Available Successful oocyte fertilization and normal embryonic development of mice were considered the most important diagnostic criteria for the safety of materials and tools used for human in vitro fertilization and embryo transfer (IVF-ET. Therefore, we studied the influence of cumulus cells co-culture and protein supplement within culture medium on percentages of in vitro fertilization (IVF and normal development of early stages of mouse embryo later. Oocytes were collected and treated with hyaluronidase to remove cumulus cells. Oocytes were divided into four groups namely: Group-1: Oocytes incubated within modified Earl’s medium (MEM supplied with 10% inactivated bovine amniotic fluid as a protein source and cumulus cells; Group-2: Oocytes incubated with MEM supplied with cumulus cells only; Group-3: Oocytes incubated with MEM supplied with 10% inactivated bovine amniotic fluid only; and Group-4: Oocytes  incubated with MEM free of both protein source and cumulus cells. For IVF, 5-6 oocytes were incubated with active spermatozoa under paraffin oil for 18-20 hours at 37° oC in 5% CO2. Percentages of IVF and embryonic development were then recorded. Best results for IVF and normal embryonic development were achieved from oocytes of Group-1 when compared to the other groups. As compared to Group-1, the percentage of IVF for Group-2 and Group-3 were decreased insignificantly and significantly (P<0.002, respectively. Significant (P<0.01 reduction in the percentages of IVF and normal embryonic development were reported in Group-4 as compared to Group-1. Therefore, it was concluded that the presence of cumulus cells co-culture and bovine amniotic fluid as a protein source within culture medium may have an important role on the fertilizing capacity of spermatozoa and oocytes and normal development of pre-implanted mouse embryo later.

  18. BMP15 Prevents Cumulus Cell Apoptosis Through CCL2 and FBN1 in Porcine Ovaries

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    Bo Zhai

    2013-07-01

    Full Text Available Background: Bone morphogenetic protein-15 (BMP15 is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. Methods: In this study, flow cytometry (FCM was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi method was used to inhibit the expression of (MCP-1/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR and western blotting. Results: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1/CCL2 and fibrillin1 (FBN1 were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. Conclusion: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.

  19. Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

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    Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

    2013-08-01

    The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

  20. Cumulus cell mitochondrial activity in relation to body mass index in women undergoing assisted reproductive therapy

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    Victoria K. Gorshinova

    2017-06-01

    Full Text Available Most studies have considered the negative influence of obesity on fertility in both genders. In the present study, we assessed mitochondrial activity expressed as the mitochondrial potential index (MPI in cumulus cells from obese women and women with a normal body mass index (BMI during assisted reproductive therapy. The results revealed a significant reduction of MPI with increased body mass. The lower MPI levels in cumulus cells from obese women may reflect mitochondrial dysfunction caused by oxidative stress, which can affect the cumulus-oocyte complex and have an impact on oocyte development.

  1. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

  2. Mitochondrial dysfunction and apoptosis in cumulus cells of type I diabetic mice.

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    Qiang Wang

    2010-12-01

    Full Text Available Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females.

  3. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

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    Shuzhen Liu

    Full Text Available Acrylamide (ACR is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

  4. Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation

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    Yi Fang

    2018-02-01

    Full Text Available Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs were collected from eighteen lambs which were randomly divided into control group (C and melatonin group given an 18 mg melatonin implant subcutaneous (M. Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p < 0.05, greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p < 0.05. Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p < 0.05. Furthermore, melatonin increased FI of FITC (global methylation level on cumulus cells (p < 0.05. To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p < 0.05 and higher methylation at two CpG sites of DNMT3a (p < 0.05, there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p < 0.05. In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs.

  5. Embryo quality predictive models based on cumulus cells gene expression

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    Devjak R

    2016-06-01

    Full Text Available Since the introduction of in vitro fertilization (IVF in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

  6. Spermatozoa of the shrew, Suncus murinus, undergo the acrosome reaction and then selectively kill cells in penetrating the cumulus oophorus.

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    Kaneko, T; Iida, H; Bedford, J M; Mōri, T

    2001-08-01

    In the musk shrew, Suncus murinus (and other shrews), the cumulus oophorus is ovulated as a discrete, compact, matrix-free ball of cells linked by specialized junctions. In examining how they penetrate the cumulus, Suncus spermatozoa were observed to first bind consistently by the ventral face over the acrosomal region to the exposed smooth surface of a peripheral cumulus cell. This was apparently followed by point fusions between the plasma and outer acrosomal membranes. Thereafter, spermatozoa without acrosomes were observed within cumulus cells that displayed signs of necrosis, as did some radially neighboring cumulus cells linked by zona adherens and gap junctions. Eventually, penetration of spermatozoa as far as the perizonal space around the zona pellucida left linear tracks of locally necrotic cells flanked by normal cumulus cells. Based on these and previous observations, we conclude that the acrosome reaction in Suncus is always induced by cumulus cells, and that reacted spermatozoa penetrate the cumulus by selective invasion and killing of cumulus cells along a linear track. Loss of the acrosome also exposes an apical body/perforatorium that is covered with barbs that appear to assist reacted fertilizing spermatozoa in binding to the zona pellucida. Because fertilized eggs displayed no other spermatozoa within or bound to the zona, an efficient block to polyspermy must prevent such binding of additional spermatozoa.

  7. The possible FAT1-mediated apoptotic pathways in porcine cumulus cells

    NARCIS (Netherlands)

    Wu, Xinhui; Fu, Yao; Liu, Chang; Chai, Menglong; Chen, Chengzhen; Dai, Lisheng; Gao, Yan; Jiang, Hao; Zhang, Jiabao

    Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles, and the development of embryos. Studies of FAT1 have demonstrated its functions in cell-cell

  8. Effects of TGF-beta and GDF-9 on cumulus expension and progesterone production by oocytectomized oocyte-cumulus cell complexes

    Czech Academy of Sciences Publication Activity Database

    Vanderhyden, B. C.; Nagyová, Eva; Dhawan, D.

    2001-01-01

    Roč. 64, č. 1 (2001), s. 56 ISSN 0006-3363. [Society for the Study of Reproduction - Annual Meeting /34./. 28.07.2001-01.08.2001, Ottawa] Institutional research plan: CEZ:AV0Z5045916 Keywords : cumulus cells * oocytes Subject RIV: EB - Genetics ; Molecular Biology

  9. Effects of Mitochondrial Uncoupling Protein 2 Inhibition by Genipin in Human Cumulus Cells

    Directory of Open Access Journals (Sweden)

    Hongshan Ge

    2015-01-01

    Full Text Available UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality.

  10. Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

    Science.gov (United States)

    Dau, Andressa Minussi Pereira; da Silva, Eduardo Pradebon; da Rosa, Paulo Roberto Antunes; Bastiani, Felipe Tusi; Gutierrez, Karina; Ilha, Gustavo Freitas; Comim, Fabio Vasconcellos; Gonçalves, Paulo Bayard Dias

    2016-07-01

    The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (Pmeiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (Pmeiosis in cattle. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Effect of glycine and alanine supplementation on development of cattle embryos cultured in CR1aa medium with or without cumulus cells

    Directory of Open Access Journals (Sweden)

    Kr. BREDBACKA

    2008-12-01

    Full Text Available The effect of alanine (1 mM and glycine (10 mM supplementation on bovine embryo development in vitro was investigated. Presumptive bovine zygotes, produced by in vitro maturation and insemination of oocytes, were cultured for 144 h in CR1aa medium in the absence (Experiments 1 and 2 or presence of cumulus cells (Experiment 3. In Experiment 1, the proportion of morulae and blastocysts of cleaved embryos in glycine-supplemented medium was not different from that of the control medium (34% in both mediaglycine-enriched medium (69.5 vs. 53.3, P = 0.016. In Experiment 2, addition of alanine did not improve the formation of morulae and blastocysts (13% vs. 21% in control medium, and the mean cell numbers in morulae and blastocysts were lower than those in the control group (34.3 vs. 68.7, P = 0.007. In the presence of cumulus cells, the combined supplementation of glycine and alanine increased the proportion of morulae and blastocysts over that in the control medium (31% vs. 14%, P = 0.003.;

  12. Aberrant expression of long noncoding RNAs in cumulus cells isolated from PCOS patients.

    Science.gov (United States)

    Huang, Xin; Hao, Cuifang; Bao, Hongchu; Wang, Meimei; Dai, Huangguan

    2016-01-01

    To describe the long noncoding RNA (lncRNA) profiles in cumulus cells isolated from polycystic ovary syndrome (PCOS) patients by employing a microarray and in-depth bioinformatics analysis. This information will help us understand the occurrence and development of PCOS. In this study, we used a microarray to describe lncRNA profiles in cumulus cells isolated from ten patients (five PCOS and five normal women). Several differentially expressed lncRNAs were chosen to validate the microarray results by quantitative RT-PCR (qRT-PCR). Then, the differentially expressed lncRNAs were classified into three subgroups (HOX loci lncRNA, enhancer-like lncRNA, and lincRNA) to deduce their potential features. Furthermore, a lncRNA/mRNA co-expression network was constructed by using the Cytoscape software (V2.8.3, http://www.cytoscape.org/ ). We observed that 623 lncRNAs and 260 messenger RNAs (mRNAs) were significantly up- or down-regulated (≥2-fold change), and these differences could be used to discriminate cumulus cells of PCOS from those of normal patients. Five differentially expressed lncRNAs (XLOC_011402, ENST00000454271, ENST00000433673, ENST00000450294, and ENST00000432431) were selected to validate the microarray results using quantitative RT-PCR (qRT-PCR). The qRT-PCR results were consistent with the microarray data. Further analysis indicated that many differentially expressed lncRNAs were transcribed from chromosome 2 and may act as enhancers to regulate their neighboring protein-coding genes. Forty-three lncRNAs and 29 mRNAs were used to construct the coding-non-coding gene co-expression network. Most pairs positively correlated, and one mRNA correlated with one or more lncRNAs. Our study is the first to determine genome-wide lncRNA expression patterns in cumulus cells isolated from PCOS patients by microarray. The results show that clusters of lncRNAs were aberrantly expressed in cumulus cells of PCOS patients compared with those of normal women, which revealed

  13. Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

    Directory of Open Access Journals (Sweden)

    Barboni Barbara

    2003-05-01

    Full Text Available Abstract Background The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s. To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM. The secretion of the factor(s in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC steroidogenesis. Methods In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s can influence GC steroidogenesis in the pig was confirmed in our lab by studying E2 and P4 production by cumulus cells monolayers using a radioimmunoassay technique. Results Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by

  14. Effect of melatonin on in vitro maturation of bovine oocytes ...

    African Journals Online (AJOL)

    ... Vs 17.67, 15.68, 16.53). In conclusion in this experiment, melatonin cannot improve cumulus cell expansion and nuclear maturation of bovine oocytes. When concentrations is high, melatonin may affect bovine oocytes meiotic maturation at metaphase-1 stage, but it is improbable melatonin be toxic for bovine oocytes.

  15. Effects of exogenous hyaluronic acid and serum on matrix organization and stability in the mouse cumulus cell-oocyte complex.

    Science.gov (United States)

    Camaioni, A; Hascall, V C; Yanagishita, M; Salustri, A

    1993-09-25

    Compact cumulus cell-oocyte complexes (COCs) isolated from preovulatory mouse follicles undergo expansion in vitro when high levels of hyaluronic acid (HA) are synthesized and organized into an extracellular matrix. We studied the effects of fetal bovine serum (FBS) and of exogenous HA and HA-oligomers on the expansion process. Maximum retention of HA in the COC matrix, and hence complete COC expansion, occurs when 1% FBS is continuously present during the first 18 h of culture. Irrespective of the culture time, HA synthesized when serum is absent is primarily in the medium, whereas HA synthesized when serum is present is primarily in the cell matrix. These findings support the hypothesis that the serum factor, identified as an inter-alpha-trypsin inhibitor by Chen et al. (Chen, L., Mao, S. J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380-12386), is a structural component of the matrix. Addition of exogenous HA or of HA oligomers of decasaccharide size (GlcUA-GlcNAc)5 or larger effectively displaces endogenously synthesized HA from the matrix into the medium, thereby preventing COC expansion. Addition of exogenous chondroitin sulfate affects neither matrix organization nor COC expansion, thus indicating specificity of the binding of some structural component(s) to HA. Fully expanded COCs disassemble when cultured longer than 18 h, a process which occurs also in vivo and which correlates with loss of oocyte fertilizability both in vivo and in vitro. This process involves release of macromolecular HA from the matrix into the medium, with loss of 50% of the HA in the first 8 h of incubation after full expansion. The release is not facilitated when HA oligomers, long enough to prevent matrix formation, are added to the culture medium after the COCs are fully expanded. This suggests that cooperative binding to HA of either the serum factor, an endogenously synthesized factor(s), or both is required to stabilize the fully expanded COC matrix.

  16. Bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

  17. Anti-Müllerian hormone remains highly expressed in human cumulus cells during the final stages of folliculogenesis

    DEFF Research Database (Denmark)

    Grøndahl, M L; Nielsen, M Eilsø; Dal Canto, M B

    2011-01-01

    This study evaluated whether anti-Müllerian hormone (AMH) was differentially expressed in cumulus (CC) and granulosa (GC) cells from large antral and pre-ovulatory follicles collected from individual follicles in women undergoing in-vitro maturation (IVM) or IVF treatment. Expression studies of A...

  18. Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications.

    Science.gov (United States)

    Da Broi, M G; Giorgi, V S I; Wang, F; Keefe, D L; Albertini, D; Navarro, P A

    2018-03-02

    An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.

  19. Factors affecting the outcome of in vitro bovine embryo production using ovum pick-up-derived cumulus oocyte complexes

    NARCIS (Netherlands)

    Merton, J.S.

    2013-01-01

    Optimization of bovine ovum pick up (OPU) followed by in vitro embryo production (IVP) has been driven by the desire of both beef and dairy cattle breeders to enhance genetic improvement. The work presented in this thesis focuses on optimizing the efficiency and efficacy of the OPU-IVP program.

  20. Comparative Gene Expression Profiling in Human Cumulus Cells according to Ovarian Gonadotropin Treatments

    Directory of Open Access Journals (Sweden)

    Said Assou

    2013-01-01

    Full Text Available In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i to characterize and compare gene expression profiles in cumulus cells (CCs of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A and cell-to-cell interactions (GJA5. Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1. Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3 were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality.

  1. Transcriptomic Analysis and Meta-Analysis of Human Granulosa and Cumulus Cells.

    Directory of Open Access Journals (Sweden)

    Tanja Burnik Papler

    Full Text Available Specific gene expression in oocytes and its surrounding cumulus (CC and granulosa (GC cells is needed for successful folliculogenesis and oocyte maturation. The aim of the present study was to compare genome-wide gene expression and biological functions of human GC and CC. Individual GC and CC were derived from 37 women undergoing IVF procedures. Gene expression analysis was performed using microarrays, followed by a meta-analysis. Results were validated using quantitative real-time PCR. There were 6029 differentially expressed genes (q < 10-4; of which 650 genes had a log2 FC ≥ 2. After the meta-analysis there were 3156 genes differentially expressed. Among these there were genes that have previously not been reported in human somatic follicular cells, like prokineticin 2 (PROK2, higher expressed in GC, and pregnancy up-regulated nonubiquitous CaM kinase (PNCK, higher expressed in CC. Pathways like inflammatory response and angiogenesis were enriched in GC, whereas in CC, cell differentiation and multicellular organismal development were among enriched pathways. In conclusion, transcriptomes of GC and CC as well as biological functions, are distinctive for each cell subpopulation. By describing novel genes like PROK2 and PNCK, expressed in GC and CC, we upgraded the existing data on human follicular biology.

  2. Competence Classification of Cumulus and Granulosa Cell Transcriptome in Embryos Matched by Morphology and Female Age.

    Directory of Open Access Journals (Sweden)

    Rehannah Borup

    Full Text Available By focussing on differences in the mural granulosa cell (MGC and cumulus cell (CC transcriptomes from follicles resulting in competent (live birth and non-competent (no pregnancy oocytes the study aims on defining a competence classifier expression profile in the two cellular compartments.A case-control study.University based facilities for clinical services and research.MGC and CC samples from 60 women undergoing IVF treatment following the long GnRH-agonist protocol were collected. Samples from 16 oocytes where live birth was achieved and 16 age- and embryo morphology matched incompetent oocytes were included in the study.MGC and CC were isolated immediately after oocyte retrieval. From the 16 competent and non-competent follicles, mRNA was extracted and expression profile generated on the Human Gene 1.0 ST Affymetrix array. Live birth prediction analysis using machine learning algorithms (support vector machines with performance estimation by leave-one-out cross validation and independent validation on an external data set.We defined a signature of 30 genes expressed in CC predictive of live birth. This live birth prediction model had an accuracy of 81%, a sensitivity of 0.83, a specificity of 0.80, a positive predictive value of 0.77, and a negative predictive value of 0.86. Receiver operating characteristic analysis found an area under the curve of 0.86, significantly greater than random chance. When applied on 3 external data sets with the end-point outcome measure of blastocyst formation, the signature resulted in 62%, 75% and 88% accuracy, respectively. The genes in the classifier are primarily connected to apoptosis and involvement in formation of extracellular matrix. We were not able to define a robust MGC classifier signature that could classify live birth with accuracy above random chance level.We have developed a cumulus cell classifier, which showed a promising performance on external data. This suggests that the gene signature at

  3. APOPTOSIS RATE IN CUMULUS CELLS AS POSSIBLE MOLECULAR BIOMARKER FOR OOCYTE COMPETENCE.

    Directory of Open Access Journals (Sweden)

    Liana Bosco

    2017-04-01

    Full Text Available Several lines of evidence showed that apoptosis rate of cumulus cells in oocytes derived by assisted reproductive technologies could be used as an indicator of fertilizing gamete quality. Aim of the study was to investigate the effects of three different ovarian stimulation protocols on the biological and clinical outcome in hyporesponder patients. Collected data showed a higher significant rate of DNA fragmentation index (DFI in U group (patients treated with Highly Purified human Menopausal Gonadotrophin than in P group (treated with recombinant human Follicle Stimulating Hormone (r-hFSH combined with recombinant human Luteinizing Hormone (r-hLH. Both groups R (treated with r-hFSH alone and P showed a significant increase in collected and fertilized oocytes number, embryo quality number. This study showed that combined r-hFSH/r-hLH therapy could represent the best pharmacological strategy for controlled ovarian stimulation and suggests to use DFI as a biomarker of ovarian function in hyporesponder patients.

  4. Female Aging Alters Expression of Human Cumulus Cells Genes that Are Essential for Oocyte Quality

    Directory of Open Access Journals (Sweden)

    Tamadir Al-Edani

    2014-01-01

    Full Text Available Impact of female aging is an important issue in human reproduction. There was a need for an extensive analysis of age impact on transcriptome profile of cumulus cells (CCs to link oocyte quality and developmental potential with patient’s age. CCs from patients of three age groups were analyzed individually using microarrays. RT-qPCR validation was performed on independent CC cohorts. We focused here on pathways affected by aging in CCs that may explain the decline of oocyte quality with age. In CCs collected from patients >37 years, angiogenic genes including ANGPTL4, LEPR, TGFBR3, and FGF2 were significantly overexpressed compared to patients of the two younger groups. In contrast genes implicated in TGF-β signaling pathway such as AMH, TGFB1, inhibin, and activin receptor were underexpressed. CCs from patients whose ages are between 31 and 36 years showed an overexpression of genes related to insulin signaling pathway such as IGFBP3, PIK3R1, and IGFBP5. A bioinformatic analysis was performed to identify the microRNAs that are potential regulators of the differentially expressed genes of the study. It revealed that the pathways impacted by age were potential targets of specific miRNAs previously identified in our CCs small RNAs sequencing.

  5. CHEMERIN (RARRES2) decreases in vitro granulosa cell steroidogenesis and blocks oocyte meiotic progression in bovine species.

    Science.gov (United States)

    Reverchon, Maxime; Bertoldo, Michael J; Ramé, Christelle; Froment, Pascal; Dupont, Joëlle

    2014-05-01

    CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation. © 2014 by the Society for the Study of Reproduction, Inc.

  6. Estudo estereológico comparativo de complexos cumulus-ovócito aspirados de folículos durante o ciclo estral em bovinos Comparative stereological study of cumulus-oocyte complexes aspirated from follicles during the estrous cycle in bovine

    Directory of Open Access Journals (Sweden)

    A.M. Calado

    2005-08-01

    Full Text Available Realizou-se uma análise estereológica comparativa de complexos cumulus-ovócito (COCs de bovino da raça Holtein-Friesian aspirados de folículos antrais pequenos (com diâmetro de 1-4mm e médios (com diâmetro de 4-8mm durante as fases de metaestro, diestro e de proestro. Foram estimados o volume médio dos COCs, dos ovócitos (com e sem zona pelúcida, dos núcleos dos ovócitos e das células foliculares e seus respectivos núcleos. Estimou-se a espessura da zona pelúcida e calculou-se a percentagem relativa da freqüência dos diferentes tipos de células foliculares encontradas no cumulus. Os folículos pequenos apresentaram crescimento acelerado e sem sincronia entre o volume do citoplasma e o do núcleo. No folículo médio ocorreu expansão harmoniosa núcleo-citoplasmática. Identificaram-se três populações de células foliculares (C1, C2 e C3, cuja distribuição na massa do cumulus é independente de sua posição relativamente ao ovócito. Durante o ciclo estral, as células C1 foram progressivamente substituídas por C2 e estas, por C3.A comparative stereological analysis was performed in cumulus-oocyte complexes from Holstein-Friesian cows, aspirated from small (with diameter of 1-4mm and medium (with diameter of 4-8mm antral follicles during metestrous, diestrous and proestrous. The mean volumes of COCs, oocytes (with and without zona pellucida, nucleus of the oocyte, as well as the volumes of the follicular cells and their nucleus were estimated. The mean thickness of the zona pellucida and the relative percentage of the three follicular cell types in the cumulus mass were also obtained. Small antral follicles had an accelerate growth without synchrony between the volume of the oocyte and the respective nucleus, while in medium antral follicles an harmonious nucleus-cytoplasmic expansion was observed. These follicular cell populations were classified in their types, C1, C2 and C3, which the distribution in the cumulus mass

  7. Research resources: comparative microRNA profiles in human corona radiata cells and cumulus oophorus cells detected by next-generation small RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Xian-Hong Tong

    Full Text Available During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs and cumulus oophorus cells (COCs, which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.

  8. NUTRIENTS AND EPIGENETICS IN BOVINE CELLS

    Science.gov (United States)

    This is a chapter for a book titled “Livestock Epigenetics” edited by Dr. Hasan Khatib and published by Wiley-Blackwell. This chapter is focused on the research development in our laboratory in the area of interaction of nutrients and genomic phonotype in bovine cells. Briefly, the Research on nutri...

  9. Increased expression of pentraxin 3 after in vivo and in vitro stimulation with gonadotropins in porcine oocyte-cumulus complexes and granulosa cells

    Czech Academy of Sciences Publication Activity Database

    Nagyová, Eva; Kalous, Jaroslav; Němcová, Lucie

    2016-01-01

    Roč. 56, č. 1 (2016), s. 29-35 ISSN 0739-7240 R&D Projects: GA ČR GA305/05/0960 Institutional support: RVO:67985904 Keywords : oocyte-cumulus complex * granulosa cells * pentraxin 3 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.644, year: 2016

  10. Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

    Science.gov (United States)

    Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

    2015-08-01

    Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

    NARCIS (Netherlands)

    Lammers, A.; Vorstenbosch, van C.J.; Erkens, J.H.F.; Smith, H.E.

    2001-01-01

    We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover. we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other

  12. Gene expression microarray profiles of cumulus cells in lean and overweight-obese polycystic ovary syndrome patients.

    Science.gov (United States)

    Kenigsberg, Shlomit; Bentov, Yaakov; Chalifa-Caspi, Vered; Potashnik, Gad; Ofir, Rivka; Birk, Ohad S

    2009-02-01

    The aim of this work was to study gene expression patterns of cultured cumulus cells from lean and overweight-obese polycystic ovary syndrome (PCOS) patients using genome-wide oligonucleotide microarray. The study included 25 patients undergoing in vitro fertilization and intra-cytoplasmic sperm injection: 12 diagnosed with PCOS and 13 matching controls. Each of the groups was subdivided into lean (body mass index (BMI) 27) subgroups. The following comparisons of gene expression data were made: lean PCOS versus lean controls, lean PCOS versus overweight PCOS, all PCOS versus all controls, overweight PCOS versus overweight controls, overweight controls versus lean controls and all overweight versus all lean. The largest number of differentially expressed genes (DEGs), with fold change (FC) |FC| >or= 1.5 and P-value lean PCOS versus lean controls comparison (487) with most of these genes being down-regulated in PCOS. The second largest group of DEGs originated from the comparison of lean PCOS versus overweight PCOS (305). The other comparisons resulted in a much smaller number of DEGs (174, 109, 125 and 12, respectively). In the comparison of lean PCOS with lean controls, most DEGs were transcription factors and components of the extracellular matrix and two pathways, Wnt/beta-catenin and mitogen-activated protein kinase. When comparing overweight PCOS with overweight controls, most DEGs were of pathways related to insulin signaling, metabolism and energy production. The finding of unique gene expression patterns in cumulus cells from the two PCOS subtypes is in agreement with other studies that have found the two to be separate entities with potentially different pathophysiologies.

  13. The Cumulus Cell Layer Protects Bovine Maturing Oocyte Against Fatty Acid-Induced Lipotoxicity

    NARCIS (Netherlands)

    Lolicato, Francesca|info:eu-repo/dai/nl/314639586; Brouwers, Jos F.|info:eu-repo/dai/nl/173812694; van de Lest, Chris H.A.|info:eu-repo/dai/nl/146063570; Wubbolts, Richard|info:eu-repo/dai/nl/181688255; Aardema, Hilde|info:eu-repo/dai/nl/304824100; Priore, Paola; Roelen, Bernard A.J.|info:eu-repo/dai/nl/109291859; Helms, J. Bernd|info:eu-repo/dai/nl/080626742; Gadella, Bart M|info:eu-repo/dai/nl/115389873

    2015-01-01

    Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report we described the effects of the three predominant fatty acids in follicular fluid

  14. Activation of Cumulus Cell SMAD2/3 and Epidermal Growth Factor Receptor Pathways Are Involved in Porcine Oocyte-Cumulus Cell Expansion and Steroidogenesis

    Czech Academy of Sciences Publication Activity Database

    Nagyová, Eva; Camaioni, A.; Scsuková, S.; Mlynarčíková, A.; Procházka, Radek; Němcová, Lucie; Salustri, A.

    2011-01-01

    Roč. 78, č. 6 (2011), s. 391-402 ISSN 1040-452X R&D Projects: GA ČR GA523/08/0111 Institutional research plan: CEZ:AV0Z50450515 Keywords : MURAL GRANULOSA -CELLS * IN-VITRO MATURATION * PREOVULATORY OVARIAN-FOLLICLES Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.532, year: 2011

  15. No specific gene expression signature in human granulosa and cumulus cells for prediction of oocyte fertilisation and embryo implantation.

    Directory of Open Access Journals (Sweden)

    Tanja Burnik Papler

    Full Text Available In human IVF procedures objective and reliable biomarkers of oocyte and embryo quality are needed in order to increase the use of single embryo transfer (SET and thus prevent multiple pregnancies. During folliculogenesis there is an intense bi-directional communication between oocyte and follicular cells. For this reason gene expression profile of follicular cells could be an important indicator and biomarker of oocyte and embryo quality. The objective of this study was to identify gene expression signature(s in human granulosa (GC and cumulus (CC cells predictive of successful embryo implantation and oocyte fertilization. Forty-one patients were included in the study and individual GC and CC samples were collected; oocytes were cultivated separately, allowing a correlation with IVF outcome and elective SET was performed. Gene expression analysis was performed using microarrays, followed by a quantitative real-time PCR validation. After statistical analysis of microarray data, there were no significantly differentially expressed genes (FDR<0,05 between non-fertilized and fertilized oocytes and non-implanted and implanted embryos in either of the cell type. Furthermore, the results of quantitative real-time PCR were in consent with microarray data as there were no significant differences in gene expression of genes selected for validation. In conclusion, we did not find biomarkers for prediction of oocyte fertilization and embryo implantation in IVF procedures in the present study.

  16. Characterization Of Bovine Adipose-Derived Stem Cells

    OpenAIRE

    Daniel Cebo

    2017-01-01

    Bovine adipose-derived stem cells were obtained from the subcutaneous abdominal adipose tissue. The cells were cultured by the modified tissue-explants method developed in our laboratory and then analyzed using optical microscopy and flow cytometry. These cells were able to replicate in our cell culture conditions. cell Flow cytometry showed that bovine adipose-derived stem cells expressed mesenchymal stem cell markers CD73 and CD90. Meanwhile haematopoietic markers CD45 and CD34 are absent f...

  17. Expression pattern of G protein‑coupled estrogen receptor 1 (GPER) in human cumulus granulosa cells (CGCs) of patients with PCOS.

    Science.gov (United States)

    Zang, Lili; Zhang, Quan; Zhou, Yi; Zhao, Yan; Lu, Linlin; Jiang, Zhou; Peng, Zhen; Zou, Shuhua

    2016-06-01

    Estradiol mediates its actions by binding to classical nuclear receptors, estrogen receptor α (ER-α) and estrogen receptor β (ER-β), and the non-classical G protein-coupled estrogen receptor 1(GPER). Several gene knockdown models have shown the importance of the receptors for growth of the oocyte and for ovulation. The aim of our study was to identify the pattern of GPER expression in human cumulus granulosa cells (CGCs) from ovarian follicles at different stages of oocyte maturation, and the differences of GPER expression between polycystic ovary syndrome (PCOS) patients and non-PCOS women. Thirty-eight cases of PCOS patients and a control group of thirty-two infertile women without PCOS were used in this study. GPER's location in CGCs was investigated by immunohistochemistry. Quantitative RT-PCR and western blot were used to identify the quantify GPER expression. Here we demonstrated that GPER was expressed in CGCs of both PCOS patients and non-PCOS women, and the expression of GPER was decreased significantly during oocyte maturation. But the expression levels of GPER in CGCs of PCOS patients and non-PCOS women were not significantly different. The data indicate that GPER may play a role during human oocyte maturation through its action in cumulus granulosa cells. AMHRIIs: anti-Mullerian hormone type II receptors; BMI: body mass index; CGCs: cumulus granulosa cells; COH: controlled ovarian hyperstimulation; E2: estradiol; EGFR: epidermal growth factor receptor; ER-α: estrogen receptor; ER-β: estrogen receptor β; FF: follicular fluid; FSH: follicle-stimulating hormone; GCs: granulosa cells; GPER: G protein-coupled estrogen receptor 1; GV: germinal vesicle; GVBD: germinal vesicle breakdown; HCG: human chorionic gonadotropin; IRS: immunoreactive score; IVF-ET: in vitro fertilization and embryo transfer; MI: metaphase I; MII: metaphase II; MAPK: mitogen-activated protein kinase; OCCCs: oocyte corona cumulus complexes; PCOS: polycystic ovarian syndrome; q

  18. Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients.

    Science.gov (United States)

    Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

    2016-12-22

    Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women.

  19. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  20. Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

    OpenAIRE

    Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

    2012-01-01

    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

  1. LH-receptor gene expression in human granulosa and cumulus cells from antral and preovulatory follicles

    DEFF Research Database (Denmark)

    Jeppesen, Janni Vikkelsø; Kristensen, Stine Gry; Nielsen, Maria Eilsø

    2012-01-01

    Context:Human granulosa cells (GC) acquire LH receptor (LHR) expression during the follicular phase of the menstrual cycle. Currently, the precise follicular stage is unknown, and specific roles of LH in the follicular development are not fully understood.Objective:Our objective was to measure LH...

  2. Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs

    Directory of Open Access Journals (Sweden)

    Yamashita Yasuhisa

    2009-12-01

    Full Text Available Abstract Objectives During ovulation, it has been shown that LH stimulus induces the expression of numerous genes via PKA, p38 MAPK, PI3K and ERK1/2 in cumulus cells and granulosa cells. Our recent study showed that EGF-like factor and its protease (TACE/ADAM17 are required for the activation of EGF receptor (EGFR, cumulus expansion and oocyte maturation of porcine cumulus-oocyte complexes (COCs. In the present study, we investigated which signaling pathways are involved in the gene expression of EGF-like factor and in Tace/Adam17 expression in cumulus cells of porcine COC during in vitro maturation. Methods Areg, Ereg, Tace/Adam17, Has2, Tnfaip6 and Ptgs2 mRNA expressions were detected in cumulus cells of porcine COCs by RT-PCR. Protein level of ERK1/2 phosphorylation in cultured cumulus cells was analyzed by westernblotting. COCs were visualized using a phase-contrast microscope. Results When COCs were cultured with FSH and LH up to 2.5 h, Areg, Ereg and Tace/Adam17 mRNA were expressed in cumulus cells of COCs. Areg, Ereg and Tace/Adam17 gene expressions were not suppressed by PI3K inhibitor (LY294002, whereas PKA inhibitor (H89, p38 MAPK inhibitor (SB203580 and MEK inhibitor (U0126 significantly suppressed these gene expressions. Phosphorylation of ERK1/2, and the gene expression of Has2, Tnfaip6 and Ptgs2 were also suppressed by H89, SB203580 and U0126, however, these negative effects were overcome by the addition of EGF to the medium, but not in the U0126 treatment group. Conclusion The results showed that PKA, p38 MAPK and ERK1/2 positively controlled the expression of EGF-like factor and TACE/ADMA17, the latter of which impacts the cumulus expansion and oocyte maturation of porcine COCs via the EGFR-ERK1/2 pathway in cumulus cells.

  3. Association between expression of cumulus expansion markers and real-time proliferation of porcine follicular granulosa cells in a primary cell culture model.

    Science.gov (United States)

    Ciesiółka, S; Budna, J; Bryja, A; Kranc, W; Chachuła, A; Dyszkiewicz-Konwińska, M; Piotrowska, H; Bukowska, D; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    Folliculogenesis is a compound process that involves both ovarian follicle growth and oocyte development, which is tightly attached to the follicular wall. During this process, cells that form the follicle structure undergo substantial morphological and molecular modifications that finally lead to differentiation and specialization of ovarian follicular cells. The differentiation of ovarian cells encompasses formation of follicle, which is composed of theca (TCs), mural granulosa (GCs), and cumulus cells (CCs). It was previously hypothesized that GCs and CCs represent undifferentiated and highly specialized follicular cells, respectively, which may have similar primordial cell origins. In this study, we investigated the expression pattern of cumulus expansion markers such as COX2, HAS2, PTX3, and TSG6 in porcine GCs during short-term, in vitro culture. We hypothesized that these genes may display an important function in GCs in relation to cellular real-time proliferation. The expression pattern of COX2, HAS2, PTX3, and TSG6 was evaluated after using RT-qPCR in relation to confocal microscopy observations of protein expression and distribution during real-time proliferation of porcine follicular GCs. The COX2 and HAS2 mRNAs were highly expressed after 120 h of in vitro culture (IVC), whereas PTX3 and TSG6 mRNAs were increased during the first 24-48 h of IVC (P less than 0.001, P less than 0.01). Conversely, all of the encoded proteins were highly expressed after 144-168 h of IVC as compared to other culture periods (P less than 0.001, P less than 0.01). When analyzing the realtime proliferation of GCs in vitro, we observed a logarithmic increase of cell proliferation between 0 h and 120 h of IVC. However, after 120-168 h of IVC, the cells reached the lag phase of proliferation. Since it is well accepted that porcine GCs undergo luteinization shortly after 24-48 h of IVC, the expression pattern of investigated genes indicated that Cox2 and Has2 are independent from

  4. Comparison of Different Vitrification Procedures on Developmental Competence of Mouse Germinal Vesicle Oocytes in the Presence or Absence of Cumulus Cells

    Directory of Open Access Journals (Sweden)

    Mojdeh Salehnia

    2009-01-01

    Full Text Available Background: An evaluation of the developmental competence of vitrified mouse germinal vesicle(GV oocytes with various equilibration and vitrification times; in the presence or absence ofcumulus cells and by comparison between the cryotop method and straws was performed.Materials and Methods: Mouse GV oocytes were considered in cumulus-denuded oocytes(CDOs and cumulus-oocyte complexes (COCs groups. Their survival and developmental rateswere studied in the following experiments: (I exposure to different equilibration times (0, 3 and5 minutes and vitrification (1, 3 and 5 minutes without plunging in LN2 as toxicity tests, (IIoocytes were vitrified using straws followed by exposure to equilibration solution for 0, 3 and 5minutes and vitrification solution for 1 and 3 minutes, and (III oocytes were vitrified by cryotopfollowing exposure to equilibration for 5 minutes and vitrification for 1 minute, respectively.Results: Maturation and developmental rates of the COCs were higher than CDOs in the nonvitrifiedgroup (p<0.05. The survival and maturation rates were low in all oocytes exposed tovitrification solution for 5 minutes (p <0.05. In vitrified CDOs and COCs using straws, the survivalrates ranged from 56.9% to 85.4% and 44.0% to 84.5%, and the maturation rates from 35.3% to56.8% and 25.8% to 56.2%, respectively; which were lower than non-vitrified samples (p <0.05.Cryotop vitrified oocytes showed higher survival, maturation and fertilization rates when comparedto straw in both CDOs and COCs (p <0.05.Conclusion: The presence of cumulus cells improves developmental competence of GV oocytesin control groups but it did not affect the vitrified group. Vitrification of mouse GV oocytes usingcryotop was more effective than straws, however both vitrification techniques did not improve thecleavage rate.

  5. Adherence of Moraxella bovis to cell cultures of bovine origin.

    Science.gov (United States)

    Annuar, B O; Wilcox, G E

    1985-09-01

    The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

  6. Expression of the bone morphogenetic protein-2 (BMP2 in the human cumulus cells as a biomarker of oocytes and embryo quality

    Directory of Open Access Journals (Sweden)

    Sirin B Demiray

    2017-01-01

    Full Text Available Background: The members of the transforming growth factor-B superfamily, as the bone morphogenetic proteins (BMPs subfamily and anti-Müllerian hormone (AMH, play a role during follicular development, and the bone morphogenetic protein-2 (BMP2, AMH, and THY1 are expressed in ovaries. Aim: This study was designed to define whether or not the expressions of these proteins in human cumulus cells (CCs can be used as predictors of the oocyte and embryo competence. Settings and Design: The study included nine female patients who were diagnosed as idiopathic infertility, aged 25–33 years (median 30 years and underwent Assisted Reproductive Technologies. Materials and Methods: The CCs from 60 oocyte–cumulus complexes obtained from the nine patients were evaluated with immunofluorescence staining in respect of BMPs, AMH and THY1 markers. The CCs surrounding the same oocytes were evaluated separately according to the oocyte and embryo quality. Statistical Analysis: Quantitative data were statistically analyzed for differences using the two-sided Mann–Whitney U test (P < 0.05. Results and Conclusions: Significant differences in immunofluorescence staining were observed in oocyte quality and embryo quality for the BMP2 only (P < 0.05. No significant differences were observed for AMH or CD90/THY1. Conclusion: These results demonstrated that there is a significant difference in the expression of BMP2 in the CCs of good quality oocytes and subsequently a good embryo.

  7. Differentiation of Bovine Spermatogonial Stem Cells into Osteoblasts

    OpenAIRE

    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-01-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 da...

  8. Propagation of bovine spermatogonial stem cells in vitro

    NARCIS (Netherlands)

    Aponte, Pedro M.; Soda, Takeshi; Teerds, Katja J.; Mizrak, S. Canan; van de Kant, Henk J. G.; de Rooij, Dirk G.

    2008-01-01

    The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that

  9. Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART

    Directory of Open Access Journals (Sweden)

    Alexandros Vithoulkas

    2017-12-01

    Conclusion(s: The investigated factors, among other substances, may be causally connected to the beneficial effect observed on embryo development. Our findings suggest that co-culture with autologous cumulus cell clusters improves the outcome of embryo culture in IVF programs.

  10. Data on endogenous bovine ovarian follicular cells peptides and small proteins obtained through Top-down High Resolution Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Valérie Labas

    2017-08-01

    Full Text Available The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017 http://dx.doi.org/10.1016/j.jprot.2017.03.027 [1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892. Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes.

  11. Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress

    NARCIS (Netherlands)

    Aardema, Hilde; van Tol, Helena T A; Wubbolts, Richard W; Brouwers, Jos F H M; Gadella, Bart M; Roelen, Bernard A J

    2017-01-01

    Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while mono-unsaturated free fatty

  12. Multilineage Potential Research of Bovine Amniotic Fluid Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuhua Gao

    2014-02-01

    Full Text Available The use of amnion and amniotic fluid (AF are abundant sources of mesenchymal stem cells (MSCs that can be harvested at low cost and do not pose ethical conflicts. In human and veterinary research, stem cells derived from these tissues are promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. This work aimed to obtain and characterize bovine amniotic fluid mesenchymal stem cells (AFMSC. The bovine AF from the amniotic cavity of pregnant gilts in the early stages of gestation (3- and 4-m-old bovine embryos was collected. AFMSCs exhibit a fibroblastic-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that AFMSCs were positive for β-integrin, CD44, CD73 and CD166, but negative for CD34, CD45. Meanwhile, AFMSCs expressed ES cell markers, such as Oct4, and when appropriately induced, are capable of differentiating into ectodermal and mesodermal lineages. This study reinforces the emerging importance of these cells as ideal tools in veterinary medicine; future studies aimed at a deeper evaluation of their immunological properties will allow a better understanding of their role in cellular therapy.

  13. Expression of uncoupling protein 1 in bovine muscle cells.

    Science.gov (United States)

    Abd Eldaim, M A; Hashimoto, O; Ohtsuki, H; Yamada, T; Murakami, M; Onda, K; Sato, R; Kanamori, Y; Qiao, Y; Tomonaga, S; Matsui, T; Funaba, M

    2016-12-01

    Uncoupling protein 1 (Ucp1) is predominantly expressed in brown/beige adipocytes in mammals. Although myogenic cells have been suggested to commit to a brown adipocyte lineage through the induction of Prdm16 expression, Prdm16 is also expressed in skeletal muscle. Thus, we examined expression of Ucp1 in bovine myogenic cells. Considering that Ucp1 is a principle molecule that induces energy expenditure in brown/beige adipocytes, expression of Ucp1 is not preferable in beef cattle because of potential decrease in energy (fattening) efficiency. The RT-PCR analyses revealed the expression of Ucp1 in the skeletal muscle of cattle; expression levels were markedly lower than those in the brown fat of calves. Immunohistochemical analyses showed that Ucp1 surrounded muscle fibers, but not adipocytes residing in skeletal muscle. Myosatellite cells cultured in myogenic medium showed an increase in the expression levels of myogenic regulatory factors ( levels were greater in cells after myogenic culture for 12 d than in those after myogenic culture for 6 d ( bovine skeletal muscle, which suggests the necessity for further studies on Ucp1-mediated energy expenditure in bovine skeletal muscle.

  14. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    International Nuclear Information System (INIS)

    Serck-Hanssen, G.; Soevik, O.

    1987-01-01

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125 I-insulin was carried out at 15 0 C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table

  15. Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist.

    Science.gov (United States)

    Devjak, Rok; Fon Tacer, Klementina; Juvan, Peter; Virant Klun, Irma; Rozman, Damjana; Vrtačnik Bokal, Eda

    2012-01-01

    In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

  16. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Crane, M.St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K. (Merck Sharp and Dohme Research Labs., Rahway, NJ (USA))

    1984-06-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G/sub 2/ phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

  17. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    International Nuclear Information System (INIS)

    Crane, M. St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K.

    1984-01-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G 2 phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed. (author)

  18. Differentiation of bovine spermatogonial stem cells into osteoblasts.

    Science.gov (United States)

    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-07-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining.

  19. Bovine annulus fibrosus cell lines isolated from intervertebral discs

    Directory of Open Access Journals (Sweden)

    Petra Kraus

    2016-12-01

    Full Text Available The adult bovine (Bos taurus intervertebral disc is primarily comprised of two major tissue types: The outer annulus fibrosus (AF and the central nucleus pulposus (NP. We isolated several primary cell lineages of passage (P 0 cells from the AF tissue omitting typically used enzymatic tissue digestion protocols. The cells grow past p10 without signs of senescence in DMEM + 10% FCS on 0.1% gelatin coated/uncoated surfaces of standard cell culture plates and survive freeze-thawing. Preliminary analysis of the AF derived cells for expression of the two structural genes Col1a1 and Col2a1 was performed by PISH recapitulating the expression observed in vivo.

  20. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-01-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

  1. Prototheca zopfii isolated from bovine mastitis induced oxidative stress and apoptosis in bovine mammary epithelial cells.

    Science.gov (United States)

    Shahid, Muhammad; Gao, Jian; Zhou, Yanan; Liu, Gang; Ali, Tariq; Deng, Youtian; Sabir, Naveed; Su, Jingliang; Han, Bo

    2017-05-09

    Bovine protothecal mastitis results in considerable economic losses worldwide. However, Prototheca zopfii induced morphological alterations and oxidative stress in bovine mammary epithelial cells (bMECs) is not comprehensively studied yet. Therefore, the aim of this current study was to investigate the P. zopfii induced pathomorphological changes, oxidative stress and apoptosis in bMECs. Oxidative stress was assessed by evaluating catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) contents and lactate dehydrogenase (LDH) activity, while ROS generation and apoptosis was measured by confocal laser scanning microscopy. The results revealed that infection of P. zopfii genotype II (GTII) significantly changed bMECs morphology, increased apoptotic rate and MDA contents at 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control group, in time-dependent manner. LDH activity and ROS generation was also increased (p < 0.01) at 12 h and 24 h. However, SOD and CAT contents in bMECs infected with GTII were decreased (p < 0.05) at 12 h, while GPx (p < 0.01), SOD (p < 0.05) and CAT (p < 0.01) levels were reduced at 24 h. In case of GTI, only CAT and GPx activities were significantly decreased when the duration prolonged to 24 h but lesser than GTII. This suggested that GTII has more devastating pathogenic effects in bMECs, and the findings of this study concluded that GTII induced apoptosis and oxidative stress in bMECs via the imbalance of oxidant and antioxidant defenses as well as the production of intracellular ROS.

  2. Bovine parvovirus uses clathrin-mediated endocytosis for cell entry.

    Science.gov (United States)

    Dudleenamjil, Enkhmart; Lin, Chin-Yo; Dredge, Devin; Murray, Byron K; Robison, Richard A; Johnson, F Brent

    2010-12-01

    Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.

  3. The Effect of Lysophosphatidic Acid during In Vitro Maturation of Bovine Oocytes: Embryonic Development and mRNA Abundances of Genes Involved in Apoptosis and Oocyte Competence

    Directory of Open Access Journals (Sweden)

    Dorota Boruszewska

    2014-01-01

    Full Text Available In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs. We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2 and of LPA receptors (LPAR 1–4 in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10−5 M for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival.

  4. Transcriptomes of bovine ovarian follicular and luteal cells

    Directory of Open Access Journals (Sweden)

    Sarah M. Romereim

    2017-02-01

    Full Text Available Affymetrix Bovine GeneChip® Gene 1.0 ST Array RNA expression analysis was performed on four somatic ovarian cell types: the granulosa cells (GCs and theca cells (TCs of the dominant follicle and the large luteal cells (LLCs and small luteal cells (SLCs of the corpus luteum. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE83524. Subsequent ANOVA determined genes that were enriched (≥2 fold more or decreased (≤−2 fold less in one cell type compared to all three other cell types, and these analyzed and filtered datasets are presented as tables. Genes that were shared in enriched expression in both follicular cell types (GCs and TCs or in both luteal cells types (LLCs and SLCs are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values is shown as a figure. These data have been further analyzed and interpreted in the companion article “Gene expression profiling of ovarian follicular and luteal cells provides insight into cellular identities and functions” (Romereim et al., 2017 [1].

  5. Culture of porcine luteal cells as a substrate for in vitro maturation of porcine cumulus oocyte complexes. Establishment and characterization

    Directory of Open Access Journals (Sweden)

    Teplitz MA

    2016-12-01

    Full Text Available The aim of this study was to establish and characterize the porcine luteal cells (PLC culture for the subsequent coculture with porcine COC. The final purpose is to promote the oocyte maturation. The PLC was established using corpora lutea obtained from slaughterhouse ovaries. Corpora lutea were dissected and luteal tissue submitted to a mechanical and enzymatic digestion with collagenase IV. The cell suspension was filtered and centrifuged and the cells obtained were diluted in 15 mL of DMEM-F12 supplemented media. Diluted cells were seeded in 3 culture flasks T25, staying in a controlled environment and changing the medium every 2 days. For the analysis and characterization, the cells were assessed by the Nile red staining to detect intracellular lipids, immunocytochemistry (ICC for 3β-hydroxy steroid dehidrogenase (3β-HSD and ELISA for P4 determination. We observed the presence of lipid intracellular droplets. Also, we observed an increase of P4 concentration at 48, 96 y 144 h of primary culture and almost all the cells were positive to the ICC evaluation for 3β-HSD, showing the steroidogenic capacity of the culture cells.

  6. Effect of cumulus-oocyte complexes (COCs) culture duration on in ...

    African Journals Online (AJOL)

    We investigated and optimized the cumulus-oocyte complexes (COCs) culture duration for pig oocyte in vitro maturation and produced a number of high-quality metaphase-II (M-II) oocytes for generation of parthenotes. The present study graded the COCs into levels A, B and C according to layers of cumulus cells, which ...

  7. Detection of bluetongue virus by using bovine endothelial cells and embryonated chicken eggs.

    OpenAIRE

    Wechsler, S J; Luedke, A J

    1991-01-01

    Two systems, inoculation of bovine endothelial cells and of embryonated chicken eggs, were compared for detection of bluetongue virus (BTV) in blood specimens from experimentally inoculated sheep. For all BTV serotypes tested, embryonated chicken eggs detected longer periods of viremia than did bovine endothelial cells, primarily by detecting BTV in samples containing lower virus concentrations.

  8. Improved detection of Bovine Viral Diarrhea Virus in Bovine lymphoid cell lines using PrimeFlow RNA assay

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

  9. Closed system for bovine oocyte vitrification

    Directory of Open Access Journals (Sweden)

    Helena Ševelová

    2012-01-01

    Full Text Available The aim of our study was to develop a vitrification carrier for bovine oocyte cryopreservation. The carrier was to be cheap enough, elementary in its construction and meet contemporary requirements for a safe closed system. In a closed system, a cell is prevented from direct exposure to liquid nitrogen, thus minimizing the risk of cross-contamination. Furthermore, two questions regarding the proper vitrification technique were resolved: if it is necessary to partially denude the oocytes before the vitrification process or whether intact cumulus oocyte complexes should be frozen; and if it is more advantageous to preheat the vitrification solutions to female body temperature (39 °C or to keep them at room temperature. Our results show that it is better to partially denude the oocytes prior to vitrification because cryopreserved intact cumulus oocyte complexes often proved dark, non-homogeneous or fragmented cytoplasm after warming, with many of them having visibly widened perivitelline spaces or fractured zonae pellucidae as a result of extensive damage during vitrification. Consequently, intact cumulus oocyte complexes showed significantly lower numbers of cleavage stage embryos on Day 3 compared to partially denuded oocytes (7.4% and 26%, respectively. On the other hand, the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions. In conclusion, results achieved with the newly developed carrier were comparable to previously published studies and therefore they could be recommended for common use.

  10. Bovine oocytes and early embryos express mRNA encoding glycerol kinase but addition of glycerol to the culture media interferes with oocyte maturation.

    Science.gov (United States)

    Okawara, Sumika; Hamano, Seizo; Tetsuka, Masafumi

    2009-04-01

    Glycerol plays multi-functional roles in cellular physiology. Other than forming the backbone molecule for glycerophospholipid and triglyceride (TG), glycerol acts as an energy substrate for glycolysis. Spermatozoa are known to utilize glycerol for energy production, but there are no reports of this in oocytes. In this study, the value of glycerol as an energy substrate for bovine oocyte maturation (Exp. 1) and the gene expression of glycerol kinase (GK), an enzyme crucial for cellular glycerol utilization, in bovine oocytes and early embryos (Exp. 2) were examined. In Exp. 1, in vitro maturation (IVM) was conducted using synthetic oviduct fluid supplemented with/without glucose (1.5 mM) and/or glycerol (1.0 mM), and maturation rate, degree of cumulus expansion, glucose consumption and lactate production by cumulus-oocyte complexes (COC) were examined. In Exp. 2, to examine the developmental expression of GK mRNA, cumulus cells, oocytes and embryos at the 2-, 8- and 16-cell, morula, expanded blastocyst and hatched blastocyst stages were obtained in separate experiments, and the expression of GK mRNA was quantified using a real-time PCR. Glycerol did not support oocyte maturation or cumulus expansion. Addition of glycerol to glucose-supplemented media significantly decreased the maturation rate. Expression of GK mRNA was very low in cumulus cells, whereas an appreciable level of the transcript was observed in the oocytes. GK mRNA was detected in embryos at all the stages examined, and its expression significantly increased at the morula stage. These results indicate that glycerol, at least at the present concentration, is not beneficial as a constituent of the medium for bovine oocyte maturation. However, the appreciable levels of GK mRNA found in the oocyte and embryo imply a physiological role for glycerol in bovine oocyte maturation and embryo development.

  11. The human cumulus--oocyte complex gene-expression profile

    Science.gov (United States)

    Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

    2006-01-01

    BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

  12. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

    Science.gov (United States)

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-12-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

  13. Global gene expression response to telomerase in bovine adrenocortical cells

    International Nuclear Information System (INIS)

    Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H.

    2005-01-01

    The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state

  14. Characterization of Bovine 5′-flanking Region during Differentiation of Mouse Embryonic Stem Cells

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    Hye-Jeong Jang

    2015-12-01

    Full Text Available Embryonic stem cells (ESCs have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181 bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181 promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

  15. Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Islam M. Saadeldin

    2017-01-01

    Full Text Available Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs compared to the conventional mouse embryonic fibroblasts (MEFs were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2, cytokeratin-8 (KRT8, and interferon tau (IFNT. The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

  16. Bovine aortic endothelial cells are susceptible to Hantaan virus infection

    International Nuclear Information System (INIS)

    Bahr, U.; Muranyi, W.; Mueller, S.; Kehm, R.; Handermann, M.; Darai, G.; Zeier, M.

    2004-01-01

    Hantavirus serotype Hantaan (HTN) is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS, lethality up to 10%). The natural host of HTN is Apodemus agrarius. Recent studies have shown that domestic animals like cattle are sporadically seropositive for hantaviruses. In the present study, the susceptibility of bovine aortic endothelial cells (BAEC) expressing α V β 3 -integrin to a HTN infection was investigated. Viral nucleocapsid protein and genomic RNA segments were detected in infected BAEC by indirect immunofluorescence assay, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The results of this study strongly support our previous observation on Puumala virus (PUU) that has been propagated efficiently in BAEC. These findings open a new window to contemplate the ecology of hantavirus infection and transmission route from animal to man

  17. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

    OpenAIRE

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-01-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic ...

  18. Comparison of gene expression patterns between porcine cumulus ...

    African Journals Online (AJOL)

    These results suggest that the aberrant of gene expression patterns detected in the oocytes of NOs compared with COCs explains their reduced quality in terms of development and maturation. In conclusion, these differentially expressed mRNAs may be involved in cellular interactions between oocytes and cumulus cells ...

  19. Transfection of bovine spermatogonial stem cells in vitro.

    Science.gov (United States)

    Tajik, P; Hoseini Pajooh, Kh; Fazle Elahi, Z; Javdani Shahedin, G; Ghasemzadeh-Nava, H

    2017-01-01

    Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (Ptransfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrier groups (Ptransfection proceeds at day four. It was concluded that lipofectamine can be used safely for direct loading exogenous DNA to SSCs particularly during the fourth day of culture.

  20. Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

    Science.gov (United States)

    Castilho, Ivana G; Dantas, Stéfani Thais Alves; Langoni, Hélio; Araújo, João P; Fernandes, Ary; Alvarenga, Fernanda C L; Maia, Leandro; Cagnini, Didier Q; Rall, Vera L M

    2017-08-01

    Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap + icaA + icaD + isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  1. Biological effect of ultraviolet radiation on cattle: bovine ocular squamous cell carcinoma

    International Nuclear Information System (INIS)

    Kopecky, K.E.; Pugh, G.W. Jr.; Hughes, D.E.; Booth, G.D.; Cheville, N.F.

    1979-01-01

    The relationship between bovine ocular squamous cell carcinoma and ultraviolet radiation was studied. Experimental procedures were devised to irradiate cattle with predetermined quantities of ultraviolet beta. Irradiation induced a preneoplastic ocular growth in one of four irradiated cattle. An epizootiologic study indicates that since 1950 the occurrence of bovine ocular squamous cell carcinoma reported at slaughter has increased. This increase was real and not due to an increase in numbers of cattle

  2. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    International Nuclear Information System (INIS)

    Doupnik, C.A.; Leikauf, G.D.

    1990-01-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with [3H]arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. [3H]arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein

  3. Bovine mammary stem cells: Cell biology meets production agriculture

    Science.gov (United States)

    Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue ...

  4. Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

    Science.gov (United States)

    Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

    2013-08-01

    Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

  5. Bovine Mastitis Resistance: Novel Quantitative Trait Loci and the Role of Bovine Mammary Epithelial Cells

    OpenAIRE

    Kurz, Jacqueline P.

    2018-01-01

    Bovine mastitis, or inflammation of the mammary gland, has substantial economic and animal welfare implications. A genetic basis for mastitis resistance traits is recognized and can be used to guide selective breeding programs. The discovery of regions of the genome associated with mastitis resistance, and knowledge of the underlying molecular mechanisms responsible, can facilitate development of efficient mastitis control and therapeutic strategies. The objectives of this dissertation resear...

  6. Interaction of bovine peripheral blood polymorphonuclear cells and Leptospira species; innate responses in the natural bovine reservoir host.

    Directory of Open Access Journals (Sweden)

    Jennifer H Wilson-Welder

    2016-07-01

    Full Text Available Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and L. interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2 was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of

  7. Visualizing Cumulus Clouds in Virtual Reality

    NARCIS (Netherlands)

    Griffith, E.J.

    2010-01-01

    This thesis focuses on interactively visualizing, and ultimately simulating, cumulus clouds both in virtual reality (VR) and with a standard desktop computer. The cumulus clouds in question are found in data sets generated by Large-Eddy Simulations (LES), which are used to simulate a small section

  8. Effect of triiodothyronine on developmental competence of bovine oocytes.

    Science.gov (United States)

    Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

    2013-09-01

    Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Cumulus Microphysics and Climate Sensitivity.

    Science.gov (United States)

    del Genio, Anthony D.; Kovari, William; Yao, Mao-Sung; Jonas, Jeffrey

    2005-07-01

    Precipitation processes in convective storms are potentially a major regulator of cloud feedback. An unresolved issue is how the partitioning of convective condensate between precipitation-size particles that fall out of updrafts and smaller particles that are detrained to form anvil clouds will change as the climate warms. Tropical Rainfall Measuring Mission (TRMM) observations of tropical oceanic convective storms indicate higher precipitation efficiency at warmer sea surface temperature (SST) but also suggest that cumulus anvil sizes, albedos, and ice water paths become insensitive to warming at high temperatures. International Satellite Cloud Climatology Project (ISCCP) data show that instantaneous cirrus and deep convective cloud fractions are positively correlated and increase with SST except at the highest temperatures, but are sensitive to variations in large-scale vertical velocity. A simple conceptual model based on a Marshall-Palmer drop size distribution, empirical terminal velocity-particle size relationships, and assumed cumulus updraft speeds reproduces the observed tendency for detrained condensate to approach a limiting value at high SST. These results suggest that the climatic behavior of observed tropical convective clouds is intermediate between the extremes required to support the thermostat and adaptive iris hypotheses.

  10. Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.

    Science.gov (United States)

    Labas, Valérie; Teixeira-Gomes, Ana-Paula; Bouguereau, Laura; Gargaros, Audrey; Spina, Lucie; Marestaing, Aurélie; Uzbekova, Svetlana

    2018-03-20

    Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

    Directory of Open Access Journals (Sweden)

    Ricardo Chaves Vilela

    2013-02-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

  12. Measuring bovine gamma delta T cell function at the site of Mycobacterium bovis infection

    Science.gov (United States)

    Bovine gamma delta T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/gamma delta T cell co-culture system to eluc...

  13. Phosphoprotein phosphatase of bovine spleen cell nuclei: physicochemical properties

    International Nuclear Information System (INIS)

    Rezyapkin, V.I.; Leonova, L.E.; Komkova, A.I.

    1986-01-01

    The physicochemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were studied. The enzyme possesses broad substrate specificity and catalyzes the dephosphorylation of phosphocasein, ATP, ADP, and p-nitrophenyl phosphate (pNPP). K/sub m/ for ATP, ADP, and pNPP are equal to 0.44, 0.43, and 1.25 mM, respectively. M/sub r/ of the enzyme, according to the data of gel filtraction of Sephadex G-75 and electrophoresis in polyacrylamide gel of various concentrations is ∼ 33,000. In electrophoresis in the presence of SDS, two protein bands with M/sub r/ 12,000 and 18,000 are detected. In the enzyme molecule, acid amino acid residues predominate; two free SH groups and two disulfide bridges are detected. Phosphoprotein phosphatase is a glycoprotein, containing ∼ 22% carbonhydrates. The protein possesses a supplementary absorption maximum at 560 nm. Ammonium molybdate is a competitive inhibitor with K/sub i/ 0.37 μM, while sodium fluoride is a noncompetitive inhibitor with K/sub i/ 1.3 mM. Incubation in the presence of 2 mM phenylmethylsulfonyl fluoride for 25 h leads to a loss of ∼ 46% of the enzymatic activity. Ammonium molybdate, sodium fluoride, and PMSF are reversible inhibitors. Modifications of the SH groups, NH 2 groups, and histidine leads to a decrease in the enzymatic activity. Incubation of phosphoprotein phosphatase with [γ- 32 P]ATP leads to the incorporation of 0.33 mole 33 P per mole of the enzyme. The mechanism of hydrolysis of the phosphodiester bond, catalyzed by the enzyme, is discussed

  14. An artificially induced follicle stimulating hormone surge at the time of human chorionic gonadotrophin administration in controlled ovarian stimulation cycles has no effect on cumulus expansion, fertilization rate, embryo quality and implantation rate

    NARCIS (Netherlands)

    Vermeiden, J. P.; Roseboom, T. J.; Goverde, A. J.; Suchartwatnachai, C.; Schoute, E.; Braat, D. D.; Schats, R.

    1997-01-01

    In the spontaneous menstrual cycle, the mid-cycle gonadotrophin surge causes maturation of the cumulus-oocyte complex, mucification of cumulus cells and expansion of the cumulus oophorus, resumption of meiosis and maturation of the cytoplasm of the oocyte. Whether this is an effect purely of

  15. Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE.

    Directory of Open Access Journals (Sweden)

    Dongseob Tark

    Full Text Available Bovine spongiform encephalopathy (BSE is a zoonotic transmissible spongiform encephalopathy (TSE thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD. Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.

  16. Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Ward, W.F.; Bauer, K.D.

    1988-01-01

    Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and an increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous [3H]thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro

  17. Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells

    International Nuclear Information System (INIS)

    Zhu, Yue; Tong, Hui-Li; Li, Shu-Feng; Yan, Yun-Qin

    2017-01-01

    Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on bovine MDSC differentiation was investigated. Western blotting, immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3 expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasm of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 h, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs. After transfection with a TCEA3-inhibiting plasmid vector for 24 h, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes the differentiation of bovine MDSCs and have implications for meat production and animal rearing. - Highlights: • Muscle-derived satellite cell differentiation is promoted by TCEA3. • TCEA3 protein was localized in the cytoplasm, but not nuclei of bovine MDSCs. • TCEA3 levels increased as myotube differentiation increased. • TCEA3 affected myotube fusion, myotube counts, and MYOG and MYH3 levels.

  18. Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

    Science.gov (United States)

    Aye, Racheal; Mwirigi, Martin Kiogora; Frey, Joachim; Pilo, Paola; Jores, Joerg; Naessens, Jan

    2015-02-07

    Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.

  19. Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

    Science.gov (United States)

    Sharma, Neelesh; Jeong, Dong Kee

    2013-01-01

    The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for

  20. Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

    DEFF Research Database (Denmark)

    Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.

    2006-01-01

    Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activation...

  1. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

    Science.gov (United States)

    Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

  2. Quantitative analysis of bone morphogenetic protein 15 (BMP15 and growth differentiation factor 9 (GDF9 gene expression in calf and adult bovine ovaries

    Directory of Open Access Journals (Sweden)

    Hayashi Ken-go

    2011-03-01

    Full Text Available Abstract Background It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15 and growth and differentiation factor 9 (GDF9 play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. Methods Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR and in situ hybridization. Immunohistochemical analysis was also performed. Results The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. Conclusions Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

  3. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  4. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

    NARCIS (Netherlands)

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3) in bovine mammary gland tissue after an intramammary challenge with

  5. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

    NARCIS (Netherlands)

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3−) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small

  6. CD335 (NKp46+ T-Cell Recruitment to the Bovine Upper Respiratory Tract during a Primary Bovine Herpesvirus-1 Infection

    Directory of Open Access Journals (Sweden)

    Rahwa A. Osman

    2017-10-01

    Full Text Available Bovine natural killer (NK cells were originally defined by the NK activation receptor CD335 [natural killer cell p46-related protein (NKp46], but following the discovery of NKp46 expression on human T-cells, the definition of conventional bovine NK cells was modified to CD335+CD3− cells. Recently, a bovine T-cell population co-expressing CD335 was identified and these non-conventional T-cells were shown to produce interferon (IFN-γ and share functional properties with both conventional NK cells and T-cells. It is not known, however, if CD335+ bovine T-cells are recruited to mucosal surfaces and what chemokines play a role in recruiting this unique T-cell subpopulation. In this study, bovine herpesvirus-1 (BHV-1, which is closely related to herpes simplex virus-1, was used to investigate bovine lymphocyte cell populations recruited to the upper respiratory tract following a primary respiratory infection. Immunohistochemical staining with individual monoclonal antibodies revealed significant (P < 0.05 recruitment of CD335+, CD3+, and CD8+ lymphocyte populations to the nasal turbinates on day 5 following primary BHV-1 infection. Dual-color immunofluorescence revealed that cells recruited to nasal turbinates were primarily T-cells that co-expressed both CD335 and CD8. This non-conventional T-cell population represented 77.5% of CD355+ cells and 89.5% of CD8+ cells recruited to nasal turbinates on day 5 post-BHV-1 infection. However, due to diffuse IFN-γ staining of nasal turbinate tissue, it was not possible to directly link increased IFN-γ production following BHV-1 infection with the recruitment of non-conventional T-cells. Transcriptional analysis revealed CCL4, CCL5, and CXCL9 gene expression was significantly (P < 0.05 upregulated in nasal turbinate tissue following BHV-1 infection. Therefore, no single chemokine was associated with recruitment of non-conventional T-cells. In conclusion, the specific recruitment of CD335+ and CD8

  7. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    C Lin

    Full Text Available Our previous studies showed that bovine respiratory syncytial virus (BRSV followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2 epithelial cells with H. somni concentrated culture supernatant (CCS stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2 and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

  8. Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum

    OpenAIRE

    Ziegler, Anja; Everett, Helen; Hamza, Eman; Garbani, Mattia; Gerber, Vinzenz; Marti, Eliane; Steinbach, Falko

    2016-01-01

    BACKGROUND: Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including hor...

  9. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Fusi, Eleonora; Baldi, Antonella; Cheli, Federica

    2008-01-01

    (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures....... In BME-UV1, spermine significantly inhibited cell proliferation (Pcultured in the presence of all amines...

  10. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM) cells in cattle naturally infected with bovine tuberculosis.

    Science.gov (United States)

    Whelan, Adam O; Villarreal-Ramos, Bernardo; Vordermeier, H Martin; Hogarth, Philip J

    2011-01-01

    Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2). Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+)IL-2(+)TNF-α(+) and IFN-γ(+) TNF-α(+) response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hi)CD45RO(+)CD62L(lo) T-effector memory (T(EM)) phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM) cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future. © 2011 Whelan et al.

  11. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM cells in cattle naturally infected with bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2. Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+IL-2(+TNF-α(+ and IFN-γ(+ TNF-α(+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hiCD45RO(+CD62L(lo T-effector memory (T(EM phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

  12. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

    Directory of Open Access Journals (Sweden)

    Jorgelina Buschiazzo

    Full Text Available Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane and cholesteryl esters (stored in lipid droplets, revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs among seasons and a dynamic organizational structure

  13. Enhanced adipogenic differentiation of bovine bone marrow-derived mesenchymal stem cells

    Science.gov (United States)

    Until now, the isolation and characterization of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimize the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three 6-month-old cattle and used for p...

  14. The risks of using allogeneic cell lines for vaccine production : The example of Bovine Neonatal Pancytopenia

    NARCIS (Netherlands)

    Benedictus, Lindert; Bell, Charlotte R

    2017-01-01

    INTRODUCTION: Bovine neonatal pancytopenia (BNP) is a hemorrhagic disease that emerged in calves across Europe in 2007. Its occurrence is attributed to immunization of the calf's mother with a vaccine produced using an allogeneic cell line. Vaccine-induced alloantibodies specific for

  15. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  16. Polyfunctional CD4 T cells in the response to bovine tuberculosis

    Science.gov (United States)

    Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) and play relevant roles in several chronic infections, including human TB and HIV. However, the assessment of this response in bovine infections was not fe...

  17. Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development

    Directory of Open Access Journals (Sweden)

    Coyral-Castel Stéphanie

    2010-03-01

    Full Text Available Abstract Background Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor in bovine ovary and its role on ovarian cells and embryo, remain however to be determined. Methods Here, we identified the adiponectin system in bovine ovarian cells and embryo using RT-PCR, immunoblotting and immunohistochemistry. Furthermore, we investigated in vitro the effects of recombinant human adiponectin (10 micro g/mL on proliferation of granulosa cells (GC measured by [3H] thymidine incorporation, progesterone and estradiol secretions measured by radioimmunoassay in the culture medium of GC, nuclear oocyte maturation and early embryo development. Results We show that the mRNAs and proteins for the adiponectin system are present in bovine ovary (small and large follicles and corpus luteum and embryo. Adiponectin, AdipoR1 and AdipoR2 were more precisely localized in oocyte, GC and theca cells. Adiponectin increased IGF-1 10(-8 M-induced GC proliferation (P Conclusions In bovine species, adiponectin decreased insulin-induced steroidogenesis and increased IGF-1-induced proliferation of cultured GC through a potential involvement of ERK1/2 MAPK pathway, whereas it did not modify oocyte maturation and embryo development in vitro.

  18. CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

    Science.gov (United States)

    Heo, Young Tae; Quan, Xiaoyuan; Xu, Yong Nan; Baek, Soonbong; Choi, Hwan; Kim, Nam-Hyung; Kim, Jongpil

    2015-02-01

    Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

  19. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  20. [Study on relationship of dose-effect and time-effect of APA microencapsulated bovine chromaffin cells on pain treatment].

    Science.gov (United States)

    Hui, Jianfeng; Li, Tao; Du, Zhi; Song, Jichang

    2011-12-01

    This study was to investigate the relationship of dose-effect and time-effect of Alginate-Polylysine-Alginate (APA) microencapsulated bovine chromaffin cells on the treatment of pain model rats. Using a rat model of painful peripheral neuropathy, the antinociceptive effects of APA microencapsulated bovine cells transplanted into the subarachnoid space was evaluated by cold allodynia test and hot hyperalgesia test. Compared with control group, the withdrawal difference with cell number 50 thousands groups, 100 thousands groups and 200 thousands groups was reduced (P APA microencapsulated bovine chromaffin cells which were transplanted to treat pain model rats, and the effective antinociception remained longer than 12 weeks.

  1. Cellular and exosome mediated molecular defense mechanism in bovine granulosa cells exposed to oxidative stress.

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    Mohammed Saeed-Zidane

    Full Text Available Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 μM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo or those released by granulosa cells without oxidative stress (NormalExo were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein, altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells

  2. Ring-like distribution of constitutive heterochromatin in bovine senescent cells.

    Science.gov (United States)

    Pichugin, Andrey; Beaujean, Nathalie; Vignon, Xavier; Vassetzky, Yegor

    2011-01-01

    Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

  3. Ring-like distribution of constitutive heterochromatin in bovine senescent cells.

    Directory of Open Access Journals (Sweden)

    Andrey Pichugin

    Full Text Available BACKGROUND: Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3 and by DAPI counterstaining. METHODS: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH. RESULTS: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. CONCLUSIONS: Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

  4. Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

    OpenAIRE

    Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

    2003-01-01

    A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

  5. Use of Rat Estrus Serum for in Vitro Maturation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    AR Rafati

    2007-04-01

    Full Text Available Introduction: Superovulation produces complications in some patients, so invitro maturation of oocytes is used to decrease or eliminate these complications and improve IVF. Moreover, IVM is used for different aspects of reproductive researches. Slaughterhouse ovaries are the main source of oocytes for IVM and IVF studies. Different media has been introduced and experimented for in vitro maturation of oocytes. Animal's serum at estrus stage contains different hormones and proteins which are essential for oocyte maturation. The aim of this study was to compare three culture media for in vitro maturation (IVM of bovine oocytes; 1(controlTCM-199, 2HCG and follicular fluid (FF and 3 antibiotic. Methods: Rat estrus serum (RSS or fetal bovine serum (FBS was added to control medium. Total of 1789 compact cumulus oocyte complexes (COCs were aspirated from ovaries of slaughtered animals. Oocytes were randomly cultured in mentioned media and incubated in 38.5◦c, 5% CO2 and 95% humidity for 24 hours. The maturation of oocytes was judged according to cumulus cell expansion or randomly orcein stained oocytes and observation of polar bodies. Results: The results showed that maturation rate was significantly higher in second and third group (90.2%, 78.7% as compared to the control group (p<0.001. There was no significant difference between second and third groups (90.2 % vs. 86.6%. Conclusion: RSS is as effective as FBS for IVM of bovine oocytes and can be used as an alternative.

  6. Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells.

    Science.gov (United States)

    Magata, F; Horiuchi, M; Miyamoto, A; Shimizu, T

    2014-08-01

    Uterine bacterial infection perturbs uterine and ovarian functions in postpartum dairy cows. Peptidoglycan (PGN) produced by gram-positive bacteria has been shown to disrupt the ovarian function in ewes. The aim of this study was to determine the effect of PGN on steroid production in bovine theca cells at different stages of follicular development. Bovine theca cells isolated from pre- and post-selection ovarian follicles (8.5mm in diameter, respectively) were cultured in vitro and challenged with PGN. Steroid production was evaluated by measuring progesterone (P4) and androstenedione (A4) concentration in culture media after 48 h or 96 h of culture. Bovine theca cells expressed PGN receptors including Toll-like receptor 2 and nucleotide-binding oligomerization domain 1 and 2. Treatment with PGN (1, 10, or 50 μg/ml) led to a decrease in P4 and A4 production by theca cells in both pre- and post-selection follicles. The mRNA expression of steroidogenic enzymes were decreased by PGN treatment. Moreover, A4 production was further suppressed when theca cells of post-selection follicles were simultaneously treated by PGN and lipopolysaccharide (0.1, 1, or 10 μg/ml). These findings indicate that bacterial toxins may act locally on ovarian steroidogenic cells and compromise follicular development in postpartum dairy cows. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Characterization of putative receptors specific for quercetin on bovine aortic smooth-muscle cells

    International Nuclear Information System (INIS)

    Yu, S.C.; Becker, C.G.

    1986-01-01

    The authors have reported that tobacco glycoprotein (TGP), rutin-bovine serum albumin conjugates (R-BSA), quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells (SMC). To investigate whether there are binding sites or receptors for these polyphenol-containing molecules on SMC, the authors have synthesized 125 I-labeled rutin-bovine serum albumin ([ 125 I]R-BSA) of high specific activity (20 Ci/mmol). SMC were isolated from a bovine thoracic aorta and maintained in Eagle's minimum essential medium with 10% calf serum in culture. These SMC at early subpassages were suspended (3-5 x 10 7 cells/ml) in phosphate-buffered saline and incubated with [ 125 I]R-BSA (10 pmol) in the presence or absence of 200-fold unlabeled R-BSA, TGP, BSA, rutin, quercetin or related polyphenols, and catecholamines. Binding of [ 125 I]R-BSA to SMC was found to be reproducible and the radioligand was displaced by R-BSA, and also by TGP, rutin, quercetin, and chlorogenic acid, but not by BSA, ellagic acid, naringin, hesperetin, dopamine, epinephrine, or isoproterenol. The binding was saturable, reversible, and pH-dependent. These results demonstrate the presence of specific binding sites for quercetinon arterial SMC

  8. Effect of exogenous progesterone on cumulus characteristics of buffalo oocytes by allowing passage of more number of sperm through cumulus but not essentially fertilization

    Directory of Open Access Journals (Sweden)

    Madhusmita Panda

    2018-03-01

    Full Text Available Objective: To understand the level of progesterone (P4 in different quality of buffalo cumulus oocyte complexes (COCs and further to evaluate the effect of exogenous P4 supplementation on maturation and subsequent developmental ability of poor quality brilliant cresyl blue (BCB- COCs. Methods: Progesterone secreted by different quality of buffalo oocytes was estimated by enzyme linked immunosorbent assay and the concentration differences were translated into P4 doses to be incorporated in the maturation medium of BCB-ve COCs followed by expression analysis of genes involved in the cumulus expansion, extracellular matrix disintegration and progesterone receptor signalling. In addition, the study also evaluated the effect of exogenous P4 on sperm-cumulus interaction. Results: More than 10-fold upregulated expression of progesterone receptor in P4 supplemented oocytes signified that P4 might be acting predominantly through this receptor. Also, exogenous P4 supplementation had significant effect on transcatheter arterial chemoembolization protease regulated by P4- progesterone receptor pathway which in turn had an important role in extracellular matrix disintegration. On the contrary, cumulus expansion genes HAS2, TNFAIP6, AREG were not altered upon P4 supplementation. Also, it was observed that P4 addition did facilitate passage of significantly more number of spermatozoa through P4 treated cumulus cells. Further, incorporation of different doses of P4 did not improve significantly the cleavage and blastocyst rates of BCB-ve COCs. Conclusions: Different qualities of buffalo COCs secrete substantially diverse levels of P4, and its supplementation has a role in oocyte maturation via modulation of cumulus characteristics but perhaps not fertilization.

  9. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

    DEFF Research Database (Denmark)

    Jensen, P L; Groendahl, M L; Beck, Helle

    2012-01-01

    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...... such as Parkinson's disease, diabetes, and spinal cord injury. One of today's challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine...... the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1-8nL per blastocyst) were isolated by micromanipulation and analysed by nano...

  10. Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability.

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    Emilie L Laurin

    Full Text Available Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12 as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.

  11. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

    Directory of Open Access Journals (Sweden)

    Erika Regina Leal de Freitas

    2011-06-01

    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  12. Bee Venom Decreases LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

    Science.gov (United States)

    Jeong, Chang Hee; Cheng, Wei Nee; Bae, Hyojin; Lee, Kyung Woo; Han, Sang Mi; Petriello, Michael C; Lee, Hong Gu; Seo, Han Geuk; Han, Sung Gu

    2017-10-28

    The world dairy industry has long been challenged by bovine mastitis, an inflammatory disease, which causes economic loss due to decreased milk production and quality. Attempts have been made to prevent or treat this disease with multiple approaches, primarily through increased abuse of antibiotics, but effective natural solutions remain elusive. Bee venom (BV) contains a variety of peptides ( e.g. , melittin) and shows multiple bioactivities, including prevention of inflammation. Thus, in the current study, it was hypothesized that BV can reduce inflammation in bovine mammary epithelial cells (MAC-T). To examine the hypothesis, cells were treated with LPS (1 μg/ml) to induce an inflammatory response and the anti-inflammatory effects of BV (2.5 and 5 μg/ml) were investigated. The cellular mechanisms of BV against LPS-induced inflammation were also investigated. Results showed that BV can attenuate expression of an inflammatory protein, COX2, and pro-inflammatory cytokines such as IL-6 and TNF-α. Activation of NF-κB, an inflammatory transcription factor, was significantly downregulated by BV in cells treated with LPS, through dephosphorylation of ERK1/2. Moreover, pretreatment of cells with BV attenuated LPS-induced production of intracellular reactive oxygen species ( e.g. , superoxide anion). These results support our hypothesis that BV can decrease LPS-induced inflammatory responses in bovine mammary epithelial cells through inhibition of oxidative stress, NF-κB, ERK1/2, and COX-2 signaling.

  13. Phenotypic, ultra-structural, and functional characterization of bovine peripheral blood dendritic cell subsets.

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    Janet J Sei

    Full Text Available Dendritic cells (DC are multi-functional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets directly ex vivo, without further in vitro manipulation. Multi-color flow cytometric analysis revealed that three DC subsets could be identified. Bovine plasmacytoid DC were phenotypically identified by a unique pattern of cell surface protein expression including CD4, exhibited an extensive endoplasmic reticulum and Golgi apparatus, efficiently internalized and degraded exogenous antigen, and were the only peripheral blood cells specialized in the production of type I IFN following activation with Toll-like receptor (TLR agonists. Conventional DC were identified by expression of a different pattern of cell surface proteins including CD11c, MHC class II, and CD80, among others, the display of extensive dendritic protrusions on their plasma membrane, expression of very high levels of MHC class II and co-stimulatory molecules, efficient internalization and degradation of exogenous antigen, and ready production of detectable levels of TNF-alpha in response to TLR activation. Our investigations also revealed a third novel DC subset that may be a precursor of conventional DC that were MHC class II+ and CD11c-. These cells exhibited a smooth plasma membrane with a rounded nucleus, produced TNF-alpha in response to TLR-activation (albeit lower than CD11c+ DC, and were the least efficient in internalization/degradation of exogenous antigen. These studies define three bovine blood DC subsets with distinct phenotypic and functional characteristics which can be analyzed during immune responses to pathogens and vaccinations of cattle.

  14. Effects of vitamin D and its metabolites on cell viability and Staphylococcus aureus invasion in bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Yue, Yuan; Hymøller, Lone; Jensen, Søren Krogh

    2017-01-01

    Vitamin D has been found have various biological effects that may be potent in preventing bovine mastitis. Two forms of vitamin D, vitamin D2 (D2) and vitamin D3 (D3), can be hydroxylated to functional metabolites in cattle. The objectives of the present study were to investigate the effects of D2...... and D3 compounds on bovine mammary epithelial cell proliferation and Staphylococcus aureus (S. aureus) invasion.. Results showed that 1,25-dihydroxyvitamin D2 have an anti-proliferation activity comparable to 1,25-dihydroxyvitamin D3, while D2 and 25-hydroxyvitamin D2 (25(OH)D2) was slightly more potent...

  15. A bovine cell line that can be infected by natural sheep scrapie prions.

    Directory of Open Access Journals (Sweden)

    Anja M Oelschlegel

    Full Text Available Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice. We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  16. Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

    Science.gov (United States)

    Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

    2016-11-01

    Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

  17. Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells

    International Nuclear Information System (INIS)

    Schmidt, A.; Buddecke, E.

    1988-01-01

    Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [ 35 S]sulfate or a combination of [ 3 H]glucosamine and [ 35 S]methionine. The purified proteoheparan sulfate had an apparent M r of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component (M r ∼30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent M r of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h

  18. Cumulus convection and the terrestrial water-vapor distribution

    Science.gov (United States)

    Donner, Leo J.

    1988-01-01

    Cumulus convection plays a significant role in determining the structure of the terrestrial water vapor field. Cumulus convection acts directly on the moisture field by condensing and precipitating water vapor and by redistributing water vapor through cumulus induced eddy circulations. The mechanisms by which cumulus convection influences the terrestrial water vapor distribution is outlined. Calculations using a theory due to Kuo is used to illustrate the mechanisms by which cumulus convection works. Understanding of these processes greatly aids the ability of researchers to interpret the seasonal and spatial distribution of atmospheric water vapor by providing information on the nature of sources and sinks and the global circulation.

  19. Inhibition of Staphylococcus aureus Invasion into Bovine Mammary Epithelial Cells by Contact with Live Lactobacillus casei

    OpenAIRE

    Bouchard, Damien S.; Rault, Lucie; Berkova, Nadia; Le Loir, Yves; Even, Sergine

    2013-01-01

    Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent...

  20. Stratocumulus to Cumulus Transition by Drizzle

    Science.gov (United States)

    Yamaguchi, Takanobu; Feingold, Graham; Kazil, Jan

    2017-10-01

    The stratocumulus to cumulus transition (SCT) is typically considered to be a slow, multiday process, caused primarily by dry air entrainment associated with overshooting cumulus, with minor influence of drizzle. This study revisits the role of drizzle in the SCT with large eddy simulations coupled with a two-moment bulk microphysics scheme that includes a budget on aerosol (Na) and cloud droplet number concentrations (Nc). We show a strong precipitation-induced modulation of the SCT by drizzle initiated in penetrative cumulus under stratocumulus. Lagrangian SCT simulations are initiated with various, moderate Na (100-250 cm-3), which produce little to no drizzle from the stratocumulus. As expected, drizzle formation in cumuli is regulated by cloud depth and Nc, with stronger dependence on cloud depth, so that, for the current case, drizzle is generated in all simulations once cumulus clouds become sufficiently deep. The drizzle generated in the cumuli washes out stratocumulus cloud water and much of the aerosol, and a cumulus state appears for approximately 10 h. With additional simulations with a fixed Nc (100 cm-3), we show that prediction of Nc is necessary for this fast SCT since it is a result of a positive feedback of collision-coalescence-induced aerosol depletion that enhances drizzle formation. A fixed Nc does not permit this feedback, and thus results in weak influence of drizzle on the SCT. Simulations with fixed droplet concentrations that bracket the time varying aerosol/drop concentrations are therefore not representative of the role of drizzle in the SCT.

  1. Proteomic analysis of bovine blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille Linnert; Grøndahl, Marie Louise; Beck, Hans Christian

    2014-01-01

    by micromanipulation. From two independent replicates, 23 proteins were identified in the blastocoel fluid while 803 proteins were identified in the remaining cell material. The proteins were grouped into categories according to their gene ontology (GO) terms by which proteins involved in cell differentiation, cell...... proliferation, development, and reproduction could be derived. Proteins classified in these categories could be candidates for further functional studies to understand pluripotency and early mammalian development....

  2. Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

    Directory of Open Access Journals (Sweden)

    Aiudi Giulio

    2004-06-01

    Full Text Available Abstract The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml, Fetal Calf Serum (FCS, precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR with specific primers. Connexin 43, cyclooxygenase-2 (COX-2 and FSH receptor (FSHr mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms.

  3. Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue.

    Science.gov (United States)

    Zhang, Xiao-Gang; Wang, Yan-Hua; Han, Cong; Hu, Shan; Wang, Li-Qiang; Hu, Jian-Hong

    2015-06-01

    Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (Pcell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. A PiggyBac mediated approach for lactoferricin gene transfer in bovine mammary epithelial stem cells for management of bovine mastitis.

    Science.gov (United States)

    Sharma, Neelesh; Huynh, Do Luong; Kim, Sung Woo; Ghosh, Mrinmoy; Sodhi, Simrinder Singh; Singh, Amit Kumar; Kim, Nam Eun; Lee, Sung Jin; Hussain, Kafil; Oh, Sung Jong; Jeong, Dong Kee

    2017-11-28

    The antibacterial and anti-inflammatory properties of lactoferricin have been ascribed to its ability to sequester essential iron. The objective of the study was to clone bovine lactoferricin ( LFcinB ) gene into PiggyBac Transposon vector, expression study in the bovine mammary epithelial stem cells (bMESCs) and also to determine the antimicrobial property of recombinant LFcinB against bovine mastitis-causing organisms. The PiggyBac-LFcinB was transfected into bMESCs by electroporation and a three fold of LFcinB secretion was observed in the transfected bMESCs medium by ELISA assay. Furthermore, the assessment of antimicrobial activity against mastitis causing pathogens Staphylococcus aureus and Escherichia coli demonstrated convincing evidence to prove strong antibacterial activity of LFcinB with 14.0±1.0 mm and 18.0±1.5 mm zone of inhibition against both organisms, respectively. The present study provides the convincing evidence to suggest the potential of PiggyBac transposon system to transfer antibacterial peptide into bMESCs or cow mammary gland and also pave the way to use bovine mammary gland as the bioreactors. Simultaneously, it also suggest toward commercial utilization of LFcinB bioreactor system in pharmaceutical industry.

  5. In Vitro toxicological effects of Fumonisin B1 and Beauvericin on bovine granulosa cells

    Directory of Open Access Journals (Sweden)

    Marco Albonico

    2015-07-01

    Full Text Available Fumonisin B1 (FB1 and beauvericin (BEA are fusariotoxins found to co-exist in food and feed commodities. The aim of this study is to evaluate the individual and combined effects of FB1 and BEA on bovine granulosa cell proliferation and steroid production. Granulosa cells (GC from small bovine follicles (1-5 mm were cultured for 48 hours in 10% fetal bovine serum followed by 48 hours in a serum-free medium containing 500 ng/ml of testosterone (as an estradiol precursor, 30 ng/ml of FSH and 30 ng/ml of IGF-I with and without FB1 (3 µM and BEA (3 µM. At the end of the experiment, the numbers of GC were determined using a Coulter counter (Beckman Coulter, USA and concentrations of progesterone and estradiol in the culture medium were determined by radioimmunoassay. FB1 and BEA, both individually and in combination, showed an inhibitory effect (P 0.05 on estradiol and progesterone production, whereas BEA (3 µM, both alone and in combination with FB1 (3 µM, was found to decrease (P < 0.001 the production of both steroids drastically. In conclusion, this in vitro study indicates that FB1 and BEA, both individually and in combination, may affect GC proliferation to different extents and shows the drastic inhibitory effects of BEA on steroid production.

  6. Exposure of mouse cumulus cell nuclei to porcine ooplasmic extract eliminates TATA box protein binding to chromatin, but has no effect on DNA methylation

    OpenAIRE

    Tong, Guo Qing; Heng, Boon Chin; Ng, Soon Chye

    2006-01-01

    Purpose: The low cloning efficiency with SCNT is due to incomplete or partial reprogramming of the donor somatic cell nuclei after microinjection into the enucleated oocyte. A possible solution may be to initiate nuclear reprogramming prior to SCNT.

  7. Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

    Science.gov (United States)

    Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

    2018-01-01

    Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and

  8. Zinc supplementation protects against cadmium accumulation and cytotoxicity in Madin-Darby bovine kidney cells.

    Directory of Open Access Journals (Sweden)

    Ding Zhang

    Full Text Available Cadmium ions (Cd2+ have been reported to accumulate in bovine tissues, although Cd2+ cytotoxicity has not been investigated thoroughly in this species. Zinc ions (Zn2+ have been shown to antagonize the toxic effects of heavy metals such as Cd2+ in some systems. The present study investigated Cd2+ cytotoxicity in Madin-Darby bovine kidney (MDBK epithelial cells, and explored whether this was modified by Zn2+. Exposure to Cd2+ led to a dose- and time-dependent increase in apoptotic cell death, with increased intracellular levels of reactive oxygen species and mitochondrial damage. Zn2+ supplementation alleviated Cd2+-induced cytotoxicity and this protective effect was more obvious when cells were exposed to a lower concentration of Cd2+ (10 μM, as compared to 50 μM Cd2+. This indicated that high levels of Cd2+ accumulation might induce irreversible damage in bovine kidney cells. Metallothioneins (MTs are metal-binding proteins that play an essential role in heavy metal ion detoxification. We found that co-exposure to Zn2+ and Cd2+ synergistically enhanced RNA and protein expression of MT-1, MT-2, and the metal-regulatory transcription factor 1 in MDBK cells. Notably, addition of Zn2+ reduced the amounts of cytosolic Cd2+ detected following MDBK exposure to 10 μM Cd2+. These findings revealed a protective role of Zn2+ in counteracting Cd2+ uptake and toxicity in MDBK cells, indicating that this approach may provide a means to protect livestock from excessive Cd2+ accumulation.

  9. Histamine Induces Bovine Rumen Epithelial Cell Inflammatory Response via NF-κB Pathway

    Directory of Open Access Journals (Sweden)

    Xudong Sun

    2017-06-01

    Full Text Available Background/Aims: Subacute ruminal acidosis (SARA is a common disease in high-producing lactating cows. Rumenitis is the initial insult of SARA and is associated with the high concentrations of histamine produced in the rumen of dairy cows during SARA. However, the exact mechanism remains unclear. The objective of the current study is to investigate whether histamine induces inflammation of rumen epithelial cells and the underlying mechanism of this process. Methods: Bovine rumen epithelial cells were cultured and treated with different concentrations of histamine and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor cultured in different pH medium (pH 7.2 or 5.5. qRT-PCR, Western-blotting, ELISA and immunocytofluorescence were used to evaluate whether histamine activated the NF-κB pathway and inflammatory cytokines. Results: The results showed that histamine significantly increased the activity of IKK β and the phosphorylation levels of IκB α, as well as upregulated the mRNA and protein expression levels of NF-κB p65 in the rumen epithelial cells cultured in neutral (pH=7.2 and acidic (pH=5.5 medium. Furthermore, histamine treatment also significantly increased the transcriptional activity of NF-κB p65. High expression and transcriptional activity of NF-κB p65 significantly increased the mRNA expressions and concentrations of inflammatory cytokines, tumor necrosis factor alpha (TNF-α, interleukin 6 (IL-6 and interleukin 1 beta (IL-1β, thereby inducing the inflammatory response in bovine rumen epithelial cells. However, inhibition of NF-κB p65 by PDTC significantly decreased the expressions and concentrations of the inflammatory cytokines induced by histamine in the rumen epithelial cells cultured in the neutral and acidic medium. Conclusion: The present data indicate that histamine induces the inflammatory response of bovine rumen epithelial cells through the NF-κB pathway.

  10. Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

    Science.gov (United States)

    Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

    2014-10-01

    In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Bovine ocular squamous cell carcinoma: UV sensitivity in lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lavin, M.F.; Jennings, P.A.; Hughes, D.J. (Queensland Univ., Brisbane (Australia))

    1982-05-01

    Increased sensitivity to UV light has been demonstrated in Phytohaemagglutinin stimulated lymphocytes from normal and tumour-bearing Hereford cattle when compared to lymphocytes from other breeds. Trypan blue exclusion and inhibition of DNA synthesis were used to determine cell viability. The results obtained from time course and radiation dose experiments demonstrate biphasic survival kinetics. This is indicative of at least two separate cell populations, exhibiting differential sensitivity to UV. The increased sensitivity to UV observed in Herefords may reflect a general sensitivity to UV or alternatively a different cellular constitution in the mitogen stimulated cultures. DNA repair synthesis, measured in the presence of hydroxyurea, was of similar levels in cell cultures from Herefords and one of the control breeds.

  12. Bovine ocular squamous cell carcinoma: UV sensitivity in lymphocytes

    International Nuclear Information System (INIS)

    Lavin, M.F.; Jennings, P.A.; Hughes, D.J.

    1982-01-01

    Increased sensitivity to UV light has been demonstrated in Phytohaemagglutinin stimulated lymphocytes from normal and tumour-bearing Hereford cattle when compared to lymphocytes from other breeds. Trypan blue exclusion and inhibition of DNA synthesis were used to determine cell viability. The results obtained from time course and radiation dose experiments demonstrate biphasic survival kinetics. This is indicative of at least two separate cell populations, exhibiting differential sensitivity to UV. The increased sensitivity to UV observed in Herefords may reflect a general sensitivity to UV or alternatively a different cellular constitution in the mitogen stimulated cultures. DNA repair synthesis, measured in the presence of hydroxyurea, was of similar levels in cell cultures from Herefords and one of the control breeds. (author)

  13. [Product safety analysis of somatic cell cloned bovine].

    Science.gov (United States)

    Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

    2010-05-01

    Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

  14. Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

    International Nuclear Information System (INIS)

    Bar, R.S.; Dake, B.L.; Spanheimer, R.G.

    1985-01-01

    The ( 35 S)glycosaminoglycans (( 35 S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with 35 SO 4 for 72 h, the ( 35 S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of ( 35 S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of ( 35 S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author)

  15. Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

    Energy Technology Data Exchange (ETDEWEB)

    Bar, R.S.; Dake, B.L.; Spanheimer, R.G.

    1985-07-01

    The (/sup 35/S)glycosaminoglycans ((/sup 35/S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with /sup 35/SO/sub 4/ for 72 h, the (/sup 35/S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of (/sup 35/S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of (/sup 35/S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author). 16 refs.

  16. Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    1986-01-05

    The effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells were examined. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner. The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP)-abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56/sup 0/C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of /sup 125/I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa.

  17. Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

    OpenAIRE

    Marsh, K. A.; Hill, S. J.

    1992-01-01

    1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relat...

  18. TGF-β1 resulting in differential microRNA expression in bovine granulosa cells.

    Science.gov (United States)

    Xu, Yefen; Niu, Jiaqiang; Xi, Guangying; Niu, Xuezhi; Wang, Yuheng; Guo, Ming; Yangzong, Qiangba; Yao, Yilong; Sizhu, Suo Lang; Tian, Jianhui

    2018-07-15

    To explore the expression profile of the cellular miRNAs in bovine ovarian granulosa cells responding to transforming growth factor-β1 (TGF-β1), the effect of TGF-β1 on cell proliferation was firstly investigated by CCK-8 method and the results showed that there was a significant inhibitory effect on bovine granulosa cell proliferation treated with 5/10 ng/mL human recombinant TGF-β1 for 24 h compared to the control (P cells stimulated with or without 10 ng/mL human recombinant TGF-β1. A total of 13,257,248 and 138,726,391 clean reads per library were obtained from TGF-β1 and control groups, respectively. There were 498 and 499 bovine-specific exist miRNAs (exist miRNAs), 627 and 570 conserved known miRNAs (known miRNAs), and 593 and 585 predicted novel miRNAs in TGF-β1 and control groups, respectively. A total of 78 miRNAs with significant differential expression, including 39 up-regulated miRNAs and 39 down-regulated miRNAs were identified in the TGF-β1 group compared with the control. Real-time quantitative PCR analyses of bta-miR-106a and bta-miR-1434-5p showed that their up-expressions were interrupted by SB431542, an inhibitor that blocks TGFβ1/Smad signaling, which supported the sequencing data. GO analysis showed involvement of the predicted genes of the differentially expressed miRNAs in a broad spectrum of cell biological processes, cell components, and molecular functions. KEGG pathway analysis of the predicted miRNA targets further indicated that these differentially expressed miRNAs are involved in various signaling pathways, such as Wnt, MAPK, and TGF-β signaling, which might be involved in follicular development. These results provide valuable information on the composition, expression, and function of miRNAs in bovine granulosa cells responding to TGF-β1, and will aid in understanding the molecular mechanisms of TGF-β1 in granulosa cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Designing bovine T cell vaccines via reverse immunology

    DEFF Research Database (Denmark)

    Nene, Vishvanath; Svitek, Nicholas; Toye, Philip

    2012-01-01

    T cell responses contribute to immunity against many intracellular infections. There is, for example, strong evidence that major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) play an essential role in mediating immunity to East Coast fever (ECF), a fatal lymph...

  20. Designing bovine T-cell vaccines via reverse immunology

    Science.gov (United States)

    T-cell responses contribute to immunity against many intra-cellular infections. There is, for example, strong evidence that major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTLs) play an essential role in mediating immunity to East Coast fever (ECF), a fatal lymphop...

  1. Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

    OpenAIRE

    Li, Robert W; Li, CongJun

    2006-01-01

    Abstract Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Dis...

  2. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li Qianqian

    2010-10-01

    Full Text Available Abstract Background Spontaneous immortalisation of cultured mammary epithelial cells (MECs is an extremely rare event, and the molecular mechanism behind spontaneous immortalisation of MECs is unclear. Here, we report the establishment of a spontaneously immortalised bovine mammary epithelial cell line (BME65Cs and the changes in gene expression associated with BME65Cs cells. Results BME65Cs cells maintain the general characteristics of normal mammary epithelial cells in morphology, karyotype and immunohistochemistry, and are accompanied by the activation of endogenous bTERT (bovine Telomerase Reverse Transcriptase and stabilisation of the telomere. Currently, BME65Cs cells have been passed for more than 220 generations, and these cells exhibit non-malignant transformation. The expression of multiple genes was investigated in BME65Cs cells, senescent BMECs (bovine MECs cells, early passage BMECs cells and MCF-7 cells (a human breast cancer cell line. In comparison with early passage BMECs cells, the expression of senescence-relevant apoptosis-related gene were significantly changed in BME65Cs cells. P16INK4a was downregulated, p53 was low expressed and Bax/Bcl-2 ratio was reversed. Moreover, a slight upregulation of the oncogene c-Myc, along with an undetectable level of breast tumor-related gene Bag-1 and TRPS-1, was observed in BME65Cs cells while these genes are all highly expressed in MCF-7. In addition, DNMT1 is upregulated in BME65Cs. These results suggest that the inhibition of both senescence and mitochondrial apoptosis signalling pathways contribute to the immortality of BME65Cs cells. The expression of p53 and p16INK4a in BME65Cs was altered in the pattern of down-regulation but not "loss", suggesting that this spontaneous immortalization is possibly initiated by other mechanism rather than gene mutation of p53 or p16INK4a. Conclusions Spontaneously immortalised BME65Cs cells maintain many characteristics of normal BMEC cells and

  3. Astaxanthin increases progesterone production in cultured bovine luteal cells.

    Science.gov (United States)

    Kamada, Hachiro; Akagi, Satoshi; Watanabe, Shinya

    2017-06-29

    Although astaxanthin (AST) is known to be a strong antioxidant, its effects on reproductive function in domestic animals have not yet been elucidated in detail. Therefore, we investigated the effects of AST on luteal cells, which produce progesterone (P4), an important hormone for maintaining pregnancy. Luteal cells were prepared by collagenase dispersion of the corpus luteum (CL). The addition of racemic AST at a low concentration (production than RR-AST. When 1 mg/kg·body weight of SS-AST derived from green algae was fed to cows for 2 weeks, its concentration in blood plasma was 10.9 nM on average, which was sufficient to expect an in vitro effect on the production of P4 in cows. These results suggested the potential of SS-AST supplements for cows to elevate luteal function.

  4. Data, Meet Compute: NASA's Cumulus Ingest Architecture

    Science.gov (United States)

    Quinn, Patrick

    2018-01-01

    NASA's Earth Observing System Data and Information System (EOSDIS) houses nearly 30PBs of critical Earth Science data and with upcoming missions is expected to balloon to between 200PBs-300PBs over the next seven years. In addition to the massive increase in data collected, researchers and application developers want more and faster access - enabling complex visualizations, long time-series analysis, and cross dataset research without needing to copy and manage massive amounts of data locally. NASA has looked to the cloud to address these needs, building its Cumulus system to manage the ingest of diverse data in a wide variety of formats into the cloud. In this talk, we look at what Cumulus is from a high level and then take a deep dive into how it manages complexity and versioning associated with multiple AWS Lambda and ECS microservices communicating through AWS Step Functions across several disparate installations

  5. Effects of concanavalin A on the progesterone production by bovine steroidogenic luteal cells in vitro.

    Science.gov (United States)

    Destro, F C; Martin, I; Landim-Alvarenga, Fdc; Ferreira, Jcp; Pate, J L

    2016-10-01

    The aim of this study was to evaluate the effects of concanavalin A (CONA) on the progesterone (P4) production by bovine steroidogenic luteal cells (LCs) in vitro. Luteal cells were collected during the mid-luteal stage (at 10-12 days following ovulation) and processed in the laboratory. Luteal cells were grown for 7 days in a humid atmosphere with 5% CO2 , with or without 10% foetal bovine serum, and were subjected to the following treatments: control: no treatment; CONA (10 μg/ml); LH (100 μg/ml); CONA + LH; LH (100 μg/ml) + prostaglandin F2α (PGF2α) (10 ng/ml); CONA + LH + PGF2α. Samples of the culture media were collected on days 1 (D1) and 7 (D7) for P4 quantification. The cells were counted on D7 of culture. Differences between treatments were considered statistically significant at p < .05. Culture in the presence of CONA decreased the P4-secreting capacity of LCs on D7 of culture, particularly in the absence of serum. The cell numbers did not change between treatments. © 2016 Blackwell Verlag GmbH.

  6. Apoptotic effects of bovine apo-lactoferrin on HeLa tumor cells.

    Science.gov (United States)

    Luzi, Carla; Brisdelli, Fabrizia; Iorio, Roberto; Bozzi, Argante; Carnicelli, Veronica; Di Giulio, Antonio; Lizzi, Anna Rita

    2017-01-01

    Lactoferrin (Lf), a cationic iron-binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron-free bovine lactoferrin (apo-bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron-free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric-bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl-2, Sirt1, Mcl-1, and PARP-1 were modulated by 1.25 μM of apo-bLf. In the same cell line, apo-bLf induced apoptosis together with poly (ADP-ribose) polymerase cleavage, caspase activation, and a significant drop of NAD + . In addition, apo-bLf-treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo-bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease. Copyright © 2017 John Wiley & Sons, Ltd.

  7. Mesenchymal stem cells can survive on the extracellular matrix-derived decellularized bovine articular cartilage scaffold

    Directory of Open Access Journals (Sweden)

    Amin Tavassoli

    2015-12-01

    Full Text Available Objective (s: The scarcity of articular cartilage defect to repair due to absence of blood vessels and tissue engineering is one of the promising approaches for cartilage regeneration. The objective of this study was to prepare an extracellular matrix derived decellularized bovine articular cartilage scaffold and investigate its interactions with seeded rat bone marrow mesenchymal stem cells (BM-MSCs. Materials and Methods: Bovine articular cartilage that was cut into pieces with 2 mm thickness, were decellularized by combination of physical and chemical methods including snap freeze-thaw and treatment with sodium dodecyl sulfate (SDS. The scaffolds were then seeded with 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (DiI labeled BM-MSCs and cultured for up to two weeks. Results: Histological studies of decellularized bovine articular cartilage showed that using 5 cycles of snap freeze-thaw in liquid nitrogen and treatment with 2.5% SDS for 4 hr led to the best decellularization, while preserving the articular cartilage structure. Adherence and penetration of seeded BM-MSCs on to the scaffold were displayed by histological and florescence examinations and also confirmed by electron microscopy. Conclusion: ECM-derived decellularized articular cartilage scaffold provides a suitable environment to support adhesion and maintenance of cultured BM-MSCs and could be applied to investigate cellular behaviors in this system and may also be useful for studies of cartilage tissue engineering.

  8. Interaction between bovine-associated coagulase-negative staphylococci species and strains and bovine mammary epithelial cells reflects differences in ecology and epidemiological behavior.

    Science.gov (United States)

    Souza, F N; Piepers, S; Della Libera, A M M P; Heinemann, M B; Cerqueira, M M O P; De Vliegher, S

    2016-04-01

    Bacteria adherence seems to be an essential first stage for the internalization of bacteria into the cytoplasm of the host cell, which is considered an important virulence strategy enabling bacteria to occupy a microenvironment separated from host defense mechanisms. Thus, this study aimed to explore the difference in the capacity of 4 bovine-associated staphylococci species or strains to adhere to and internalize into bovine mammary epithelial cells (MEC). Three different isolates of coagulase-negative staphylococci (CNS) were used: one strain of Staphylococcus fleurettii isolated from sawdust and considered an environmental opportunistic bacterium, and 2 dissimilar Staphylococcus chromogenes isolates, one cultured from a heifer's teat apex (Staph. chromogenes TA) and the other originating from a chronic intramammary infection (Staph. chromogenes IM). Also, one well-characterized strain of Staphylococcus aureus (Newbould 305) was used for comparison with a major mastitis pathogen. The CNS species and strains adhered to and internalized into MEC slower than did Staph. aureus. Still, we observed high variation in adhesion and internalization capacity among the different CNS, with Staph. chromogenes IM showing a greater ability to adhere to and internalize into MEC than the 2 CNS strains isolated from extramammary habitats. In conclusion, the 3 well-characterized bovine-associated CNS species and strains originating from distinct habitats showed clear differences in their capacity to adhere to and internalize into MEC. The observed differences might be related to their diversity in ecology and epidemiological behavior. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. The role of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of bovine endometrial cell functions.

    Science.gov (United States)

    Mishra, Birendra; Kizaki, Keiichiro; Sato, Takashi; Ito, Akira; Hashizume, Kazuyoshi

    2012-06-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

  10. Alpha-Tocopherol alters transcription activities that modulate tumor necrosis factor alpha (TNF-¿)-induced inflammatory response in bovine cells

    Science.gov (United States)

    To further investigate the potential role of '-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-a as an immuno-stimulant to simulate inflammation response in cells with and without '...

  11. Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

    Science.gov (United States)

    Wang, Kai; Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

    2016-01-01

    Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

  12. Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2016-01-01

    Full Text Available Chinese propolis (CP, an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T, we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS, heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

  13. Bovine Lhx8, a Germ Cell-Specific Nuclear Factor, Interacts with Figla.

    Directory of Open Access Journals (Sweden)

    Liyuan Fu

    Full Text Available LIM homeobox 8 (Lhx8 is a germ cell-specific transcription factor essential for the development of oocytes during early oogenesis. In mice, Lhx8 deficiency causes postnatal oocyte loss and affects the expression of many oocyte-specific genes. The aims of this study were to characterize the bovine Lhx8 gene, determine its mRNA expression during oocyte development and early embryogenesis, and evaluate its interactions with other oocyte-specific transcription factors. The bovine Lhx8 gene encodes a protein of 377 amino acids. A splice variant of Lhx8 (Lhx8_v1 was also identified. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in Lhx8_v1 is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 transcripts were only detected in the gonads but none of the somatic tissues examined. The expression of Lhx8 and Lhx8_v1 appears to be restricted to oocytes as none of the transcripts was detectable in granulosa or theca cells. The maternal Lhx8 transcript is abundant in GV and MII stage oocytes as well as in early embryos but disappear by morula stage. A nuclear localization signal that is required for the import of Lhx8 into nucleus was identified, and Lhx8 is predominantly localized in the nucleus when ectopically expressed in mammalian cells. Finally, a novel interaction between Lhx8 and Figla, another transcription factor essential for oogenesis, was detected. The results provide new information for studying the mechanisms of action for Lhx8 in oocyte development and early embryogenesis.

  14. Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

    Science.gov (United States)

    Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B

    2016-04-01

    Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P gap junctions between

  15. Bovine colostrum modulates immune activation cascades in human peripheral blood mononuclear cells in vitro

    DEFF Research Database (Denmark)

    Jenny, Marcel; Pedersen, Ninfa R; Hidayat, Budi J

    2010-01-01

    factors and has a long history of use in traditional medicine. In an approach to evaluate the effects of bovine colostrum (BC) on the T-cell/macrophage interplay, we investigated and compared the capacity of BC containing low and high amounts of lactose and lactoferrin to modulate tryptophan degradation...... of lactose present in BC seems to diminish the activity of BC in our test system, since BC with higher amounts of lactose attenuated the stimulatory as well as the suppressive activity of BC....

  16. Heterogeneity in cytokine profiles of Babesia bovis-specific bovine CD4+ T cells clones activated in vitro.

    OpenAIRE

    Brown, W C; Woods, V M; Dobbelaere, D A; Logan, K S

    1993-01-01

    The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen we...

  17. Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

    International Nuclear Information System (INIS)

    Rauner, Gat; Barash, Itamar

    2014-01-01

    The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth

  18. Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rauner, Gat, E-mail: gat.rauner@mail.huji.ac.il [Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, Bet-Dagan, 50250 (Israel); The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem (Israel); Barash, Itamar, E-mail: itamar.barash@mail.huji.ac.il [Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, Bet-Dagan, 50250 (Israel)

    2014-10-15

    The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth.

  19. Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells.

    Science.gov (United States)

    Maillard, Virginie; Desmarchais, Alice; Durcin, Maeva; Uzbekova, Svetlana; Elis, Sebastien

    2018-04-26

    Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers. The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4). DHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion. These data show that DHA stimulated proliferation and steroidogenesis of bovine

  20. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhangyong1956@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China); Gao, Ming-Qing, E-mail: gaomingqing@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China)

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  1. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

    Science.gov (United States)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    International Nuclear Information System (INIS)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

    2016-01-01

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  3. Inhibition of Staphylococcus aureus invasion into bovine mammary epithelial cells by contact with live Lactobacillus casei.

    Science.gov (United States)

    Bouchard, Damien S; Rault, Lucie; Berkova, Nadia; Le Loir, Yves; Even, Sergine

    2013-02-01

    Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent invasion of bMEC by two S. aureus bovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively. L. casei strains affected adhesion and/or internalization of S. aureus in a strain-dependent manner. Interestingly, L. casei CIRM-BIA 667 reduced S. aureus Newbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two other L. casei strains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate of S. aureus was not affected by L. casei. It should be noted that L. casei was internalized at a low rate but survived in bMEC cells with a better efficiency than that of S. aureus RF122. Inhibition of S. aureus adhesion was maintained with heat-killed L. casei, whereas contact between live L. casei and S. aureus or bMEC was required to prevent S. aureus internalization. This first study of the antagonism of LAB toward S. aureus in a mammary context opens avenues for the development of novel control strategies against this major pathogen.

  4. Inhibition of Staphylococcus aureus Invasion into Bovine Mammary Epithelial Cells by Contact with Live Lactobacillus casei

    Science.gov (United States)

    Bouchard, Damien S.; Rault, Lucie; Berkova, Nadia; Le Loir, Yves

    2013-01-01

    Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent invasion of bMEC by two S. aureus bovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively. L. casei strains affected adhesion and/or internalization of S. aureus in a strain-dependent manner. Interestingly, L. casei CIRM-BIA 667 reduced S. aureus Newbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two other L. casei strains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate of S. aureus was not affected by L. casei. It should be noted that L. casei was internalized at a low rate but survived in bMEC cells with a better efficiency than that of S. aureus RF122. Inhibition of S. aureus adhesion was maintained with heat-killed L. casei, whereas contact between live L. casei and S. aureus or bMEC was required to prevent S. aureus internalization. This first study of the antagonism of LAB toward S. aureus in a mammary context opens avenues for the development of novel control strategies against this major pathogen. PMID:23183972

  5. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification.

    Science.gov (United States)

    Lai, D; Ding, J; Smith, G W; Smith, G D; Takayama, S

    2015-01-01

    Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less

  6. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...

  7. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    Science.gov (United States)

    Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

    2015-01-01

    A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

  8. Evidence of heterogeneity within bovine satellite cells isolated from young and adult animals.

    Science.gov (United States)

    Li, J; Gonzalez, J M; Walker, D K; Hersom, M J; Ealy, A D; Johnson, S E

    2011-06-01

    Satellite cells are a heterogeneous population of myogenic precursors responsible for muscle growth and repair in mammals. The objectives of the experiment were to examine the growth rates and degree of heterogeneity within bovine satellite cells (BSC) isolated from young and adult animals. The BSC were harvested from the semimembranosus of young (4.3 ± 0.5 d) and adult (estimated 24 to 27 mo) cattle and cultured en masse. Young animal BSC re-enter the cell cycle sooner and reach maximal 5-ethynyl-2'-deoxyuridine (EdU) incorporation earlier (P animals after 3, 4, and 5 d in culture. These results indicate that BSC from young animals activate, proliferate, and differentiate sooner than isolates from adult animals. Lineage heterogeneity within BSC was examined using antibodies specific for Pax7 and Myf5, lineage markers of satellite cells, and myoblasts. Immunocytochemistry revealed the majority of Pax7-expressing BSC also express Myf5; a minor population (~5%) fails to exhibit Myf5 immunoreactivity. The percentage of Pax7:Myf5 BSC from young animals decreases sooner (P cell clones were established and analyzed after 10 d. Colonies segregated into 2 groups based upon population doubling time. Immunostaining of the slow-growing colonies (population doubling time ≥ 3 d) revealed that a portion exhibited asymmetric distribution of the lineage markers Pax7 and Myf5, similar to self-renewable mouse muscle stem cells. In summary, these results offer insight into the heterogeneity of BSC and provide evidence for subtle differences between rodent and bovine myogenic precursors.

  9. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

    Science.gov (United States)

    Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

    2016-08-02

    Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

  10. Effect of Collection Technique on Yield of Bovine Oocytes and the Development Potential of Oocytes from Different Grades of Oocytes

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    R.G Sianturi

    2002-10-01

    Full Text Available Oocyte collection technique is important to obtain a maximum number of oocytes to be employed on in vitro production of embryos. In this study, immature bovine oocytes were collected from slaughterhouse ovaries by two techniques: aspiration of 2- to 6-mm follicles and slicing. Following collection, oocyte qualities were classified into four categories (A, B, C, and D on the basis of cumulus attachment. Oocytes of each category were matured in vitro in CO2 incubator for 22-24 hours and cumulus expansion and maturation rates were observed. The total number of oocytes (group A+B+C+D and yield of good quality oocytes (only group A and B recovered per ovary by aspiration were 12.02 and 8.21, and by slicing were 29.38 and 19.65 (P<0.01, respectively. The total cumulus cells expansion rates of A, B, C and D oocytes were 97.1%, 88.3%, 6.0% and 20.6% respectively. Maturation rates for A, B and C categories of oocytes were 91.4%, 82.3% and 35.0% respectively while no matured oocyte was observed for group D oocytes. Maturation rates were significantly different between group A and C and also between B and C but not between A and B (P<0.05. In conclusion, slicing technique recovered more oocytes per ovary (2.4 times than that of aspiration and the best maturation rate was observed from category A oocytes which surrounded by more than 3 layers of cumulus cells. However oocytes of category A and B can be considered as good quality oocytes.

  11. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    International Nuclear Information System (INIS)

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-01-01

    Incubation of [ 35 S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35 S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [ 35 S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [ 35 S]heparin proteoglycan after 24 hours and the appearance of free [ 35 S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [ 35 S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

  12. Bovine lactoferrin regulates cell survival, apoptosis and inflammation in intestinal epithelial cells and preterm pig intestine.

    Science.gov (United States)

    Nguyen, Duc Ninh; Jiang, Pingping; Stensballe, Allan; Bendixen, Emøke; Sangild, Per T; Chatterton, Dereck E W

    2016-04-29

    Bovine lactoferrin (bLF) may modulate neonatal intestinal inflammation. Previous studies in intestinal epithelial cells (IECs) indicated that moderate bLF doses enhance proliferation whereas high doses trigger inflammation. To further elucidate cellular mechanisms, we profiled the porcine IEC proteome after stimulation with bLF at 0, 0.1, 1 and 10g/L by LC-MS-based proteomics. Key pathways were analyzed in the intestine of formula-fed preterm pigs with and without supplementation of 10g/L bLF. Levels of 123 IEC proteins were altered by bLF. Low bLF doses (0.1-1g/L) up-regulated 11 proteins associated with glycolysis, energy metabolism and protein synthesis, indicating support of cell survival. In contrast, a high bLF dose (10g/L) up-regulated three apoptosis-inducing proteins, down-regulated five anti-apoptotic and proliferation-inducing proteins and 15 proteins related to energy and amino acid metabolism, and altered three proteins enhancing the hypoxia inducible factor-1 (HIF-1) pathway. In the preterm pig intestine, bLF at 10g/L decreased villus height/crypt depth ratio and up-regulated the Bax/Bcl-2 ratio and HIF-1α, indicating elevated intestinal apoptosis and inflammation. In conclusion, bLF dose-dependently affects IECs via metabolic, apoptotic and inflammatory pathways. It is important to select an appropriate dose when feeding neonates with bLF to avoid detrimental effects exerted by excessive doses. The present work elucidates dose-dependent effects of bLF on the proteomic changes of IECs in vitro supplemented with data from a preterm pig study confirming detrimental effects of enteral feeding with the highest dose of bLF (10g/L). The study contributes to further understanding on mechanisms that bLF, as an important milk protein, can regulate the homeostasis of the immature intestine. Results from this study urge neonatologists to carefully consider the dose of bLF to supplement into infant formula used for preterm neonates. Copyright © 2016 Elsevier B

  13. Effect of anabolics on bovine granulosa-luteal cell primary cultures.

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    Bartolomeo Biolatti

    2007-10-01

    Full Text Available Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione. Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

  14. Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications

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    DH Rosenzweig

    2017-03-01

    Full Text Available Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

  15. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

    KAUST Repository

    Janjanam, Jagadeesh

    2013-10-01

    Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Histamine Induces Bovine Rumen Epithelial Cell Inflammatory Response via NF-κB Pathway.

    Science.gov (United States)

    Sun, Xudong; Yuan, Xue; Chen, Liang; Wang, Tingting; Wang, Zhe; Sun, Guoquan; Li, Xiaobing; Li, Xinwei; Liu, Guowen

    2017-01-01

    Subacute ruminal acidosis (SARA) is a common disease in high-producing lactating cows. Rumenitis is the initial insult of SARA and is associated with the high concentrations of histamine produced in the rumen of dairy cows during SARA. However, the exact mechanism remains unclear. The objective of the current study is to investigate whether histamine induces inflammation of rumen epithelial cells and the underlying mechanism of this process. Bovine rumen epithelial cells were cultured and treated with different concentrations of histamine and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) cultured in different pH medium (pH 7.2 or 5.5). qRT-PCR, Western-blotting, ELISA and immunocytofluorescence were used to evaluate whether histamine activated the NF-κB pathway and inflammatory cytokines. The results showed that histamine significantly increased the activity of IKK β and the phosphorylation levels of IκB α, as well as upregulated the mRNA and protein expression levels of NF-κB p65 in the rumen epithelial cells cultured in neutral (pH=7.2) and acidic (pH=5.5) medium. Furthermore, histamine treatment also significantly increased the transcriptional activity of NF-κB p65. High expression and transcriptional activity of NF-κB p65 significantly increased the mRNA expressions and concentrations of inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1β), thereby inducing the inflammatory response in bovine rumen epithelial cells. However, inhibition of NF-κB p65 by PDTC significantly decreased the expressions and concentrations of the inflammatory cytokines induced by histamine in the rumen epithelial cells cultured in the neutral and acidic medium. The present data indicate that histamine induces the inflammatory response of bovine rumen epithelial cells through the NF-κB pathway. © 2017 The Author(s). Published by S. Karger AG, Basel.

  17. Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

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    Griffiths T Daniel

    2009-05-01

    Full Text Available Abstract Background The Retinal Pigmented Epithelium (RPE is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD, Retinitis Pigmentosa (RP, Fundus Flavimaculatus (FFM, Best's disease and Age-related Macular Degeneration (AMD. We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. Results The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. Conclusion The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal

  18. The unusual state of the cumulus oophorus and of sperm behaviour within it, in the musk shrew, Suncus murinus.

    Science.gov (United States)

    Bedford, J M; Mori, T; Oda, S

    1997-05-01

    In the musk shrew, Suncus murinus, the behaviour of the cumulus-egg complex and its interaction with spermatozoa were unusual in several respects. The cumulus oophorus was ovulated about 15.5 h after mating or treatment with hCG as a hyaluronidase-insensitive matrix-free ball of cells which remained for relatively long periods of about 14 h around fertilized, and for about 24 h around unfertilized eggs. As a probable function of the small number of up to about 10 or 20 spermatozoa that generally reached the oviduct ampulla from isthmic crypts, there was often a delay of up to 10 h after ovulation before most eggs were penetrated. Soon after ovulation, however, the corona radiata retreated progressively from the zona pellucida, creating a closed perizonal space within the cumulus oophorus. Usually, most spermatozoa that did reach the ampulla were found within a cumulus and generally within that perizonal space. However, whereas the acrosome was intact among the few free ampullary spermatozoa, and in those adhering to the zona of cumulus-free eggs after delayed mating, all spermatozoa seen moving within the cumulus or adhering to the zona of unfertilized eggs had shed the giant acrosome. In accord with current observations in other shrews, the cumulus in Suncus may therefore function not only to sequester spermatozoa, but also as an essential mediator of fertilization-probably by inducing the acrosome reaction. In the absence of the acrosomal carapace that expresses the zona receptors in most mammals, fertilizing Suncus spermatozoa could use an unusual array of barbs on the exposed perforatorium to attach to the zona pellucida.

  19. The effect of antibody on the adherence of Staphylococcus aureus to bovine mammary epithelial cells

    International Nuclear Information System (INIS)

    Olmsted, S.B.

    1989-01-01

    The ability of S. aureus to adhere to epithelial cells in the ductuals and alveoli of the gland is believed to add greatly to its virulence and may be necessary for colonization. Two in vitro methods were developed for the purpose of quantifying adherence. Both methods utilize bovine mammary epithelial primary cells as targets for labeled bacteria. In one assay, the bacterial are labeled with [methyl- 3 H] thymidine, and incubated on the primary epithelial monolayers. Adherence of the bacterial sample is expressed as the percent radioactivity in the adherent fraction of the total radioactivity in both fractions. The second assay involves labeling the bacteria with biotin. An enzyme-linked immunosorbent assay (ELISA) is then performed with strepavidin conjugated to horseradish peroxidase. Both methods have proven to be reliable, and allow for the testing of many criteria in one assay

  20. Cell patterning without chemical surface modification: Cell cell interactions between printed bovine aortic endothelial cells (BAEC) on a homogeneous cell-adherent hydrogel

    Science.gov (United States)

    Chen, C. Y.; Barron, J. A.; Ringeisen, B. R.

    2006-10-01

    Cell printing offers the unique ability to directly deposit one or multiple cell types directly onto a surface without the need to chemically pre-treat the surface with lithographic methods. We utilize biological laser printing (BioLP ™) to form patterns of bovine aortic endothelial cells (BAECs) onto a homogeneous cell adherent hydrogel surface. These normal cells are shown to retain near-100% viability post-printing. In order to determine whether BAECs encountered shear and/or heat stress during printing, immunocytochemical staining experiments were performed to detect potential expression of heat shock proteins (HSP) by the deposited cells. Printed BAECs expressed HSP at levels similar to negative control cells, indicating that the BioLP process does not expose cells to damaging levels of stress. However, HSP expression was slightly higher at the highest laser energy studied, suggesting more stress was present under these extreme conditions. Printed BAECs also showed preferential asymmetric growth and migration towards each other and away from the originally printed pattern, demonstrating a retained ability for the cells to communicate post-printing.

  1. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

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    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  2. Mobilisation of store Ca2+ activates tyrosine hydroxylase in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    McKenzie, S.; Marley, P.D.

    2001-01-01

    Full text: Many receptor agonists are able to activate tyrosine hydroxylase (TOH) in bovine adrenal chromaffin cells. The majority of these are dependent on extracellular Ca 2+ for this action. Entry of extracellular Ca 2+ through voltage-operated Ca 2+ channels is very effective at activating TOH. The contribution of the intracellular Ca 2+ stores to TOH activation however is not known. Previous studies have shown that mobilisation of intracellular Ca 2+ stores is effective at increasing phosphorylation of TOH, but its effect on TOH activity has not been studied. Therefore, in the present study, the effect of mobilisation of store Ca 2+ on TOH activity was investigated using primary cultures of bovine adrenal chromaffin cells. Cells were prepared from abattoir tissue and cultured for 3-6 days. TOH activity was determined over 10 minutes, measuring the 14 CO 2 produced following the hydroxylation and rapid decarboxylation of 14 C-tyrosine offered to intact cells. Caffeine increased TOH activity in a concentration-dependent manner with a maximum response of 100% increase at 20mM. This effect was not due to osmolarity since 20mM sucrose had no effect.Nor was it due to inhibition of phosphodiesterases, since the effect of caffeine was still seen in the presence of 1mM IBMX. However,caffeine-induced TOH activation was substantially reduced in the absence of extracellular Ca 2+ . The results suggest that TOH activity can be increased by mobilising intracellular Ca 2+ stores, but that this effect involves extracellular Ca 2+ influx, possibly through store-operated channels. Copyright (2001) Australian Neuroscience Society

  3. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    Science.gov (United States)

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

    2014-08-01

    Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, pvitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, pVitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

  5. Effect of loop structure of bovine lactoferricin on apoptosis in Jurkat cells.

    Science.gov (United States)

    Zhang, Tie-nan; Yang, Wei; Liu, Ning

    2010-06-01

    Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces apoptosis in Jurkat cells. However less is known about the influence of this kind of apoptosis on the intra-cellular ceramide metabolism and the structure-function relationship between the loop structure of LfcinB and its action of inducing apoptosis in Jurkat cells. In the present study, the artificially synthesized LfcinB and LfcinB-derived peptide (Cys 19 residue in LfcinB was replaced by Ala) was added in Jurkat cells, the nucleolus shape was observed by fluorescent microscopy, the ceramide concentration in Jurkat cells was determined by reversed phase high performance liquid chromatography (RP-HPLC). The results of MTT assay showed that LfcinB inhibited proliferation of Jurkat cells, and the inhibition rate was approximately 18.90%. Moreover, the inhibition rate of LfcinB together with MAPP was upto approximately 59.89%. The RP-HPLC result showed that LfcinB improved the ceramide level in Jurkat cells. By using the DNA fragmentation assay and observing the nucleolus shape, the result displayed deficiency of the loop structure could cause LfcinB losing the biological activity of inducing apoptosis in Jurkat cells.

  6. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  7. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System.

    Science.gov (United States)

    Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

    2013-01-01

    Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  8. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

    Directory of Open Access Journals (Sweden)

    Babak Qasemi-Panahi

    2013-02-01

    Full Text Available Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1 on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 μL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells.

  9. Bovine lactoferricin P13 triggers ROS-mediated caspase-dependent apoptosis in SMMC7721 cells.

    Science.gov (United States)

    Meng, Lixiang; Xu, Geliang; Li, Jiansheng; Liu, Wenbin; Jia, Weidong; Ma, Jinliang; Wei, Decheng

    2017-01-01

    Bovine lactoferricin P13 (LfcinB-P13) is a peptide derived from LfcinB. In the present study, the effect of LfcinB-P13 on the human liver cancer cell line SMMC7721 was investigated in vitro and in vivo . The results of the present study indicate that LfcinB-P13 significantly decreased SMMC7721 cell viability in vitro (P=0.032 vs. untreated cells), while exhibiting low cytotoxicity in the wild-type liver cell line L02. In addition, the rate of apoptosis in SMMC7721 cells was significantly increased following treatment with 40 and 60 µg/ml LfcinB-P13 (P=0.0053 vs. the control group), which was associated with an increase in the level of reactive oxygen species (ROS) and the activation of caspase-3 and -9. Furthermore, ROS chelation led to the suppression of LfcinB-P13-mediated caspase-3 and -9 activation in SMMC7721 cells. LfcinB-P13 was demonstrated to markedly inhibit tumor growth in an SMMC7721-xenograft nude mouse model. The results of the present study indicate that LfcinB-P13 is a novel candidate therapeutic agent for the treatment of liver cancer.

  10. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

    Science.gov (United States)

    Bieback, Karen

    2013-10-01

    Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.

  11. Evidence for alternative pathways of granulosa cell death in healthy and slightly atretic bovine antral follicles.

    Science.gov (United States)

    Van Wezel, I L; Dharmarajan, A M; Lavranos, T C; Rodgers, R J

    1999-06-01

    Granulosa cell death is an early feature of atresia; however, there are many apparent contradictions in the literature concerning the mode of granulosa cell death. We have therefore examined this process in bovine healthy and atretic antral follicles, using a variety of established techniques. Light and electron microscopic observations indicated the presence of pyknotic or shrunken nuclei in both the membrana granulosa and the antrum. In the membrana granulosa, these nuclei were frequently crescent shaped and uniformly electron dense and were approximately the same size as healthy nuclei, all of which are typical of early apoptosis. However, these nuclei were within the membranes of a healthy granulosa cell, suggesting that phagocytosis by a neighboring granulosa cell is an unusually early event in the apoptotic pathway of granulosa cells. In the membrana granulosa, pyknotic nuclei stained intensely with hematoxylin but weakly with the DNA-intercalating stain propidium iodide. A percentage of these pyknotic nuclei stained by TUNEL (terminal deoxy-UTP nick end-labeling). However, in the antrum, the pyknotic nuclei and larger globules of DNA stained intensely with both hematoxylin and propidium iodide, but were not TUNEL positive. The comet assay of cell death produced a streak tail of randomly nicked DNA, rather than the plume of low mol wt apoptotic DNA. Globules collected from fresh follicular fluid stained intensely with propidium iodide and were shown by PAGE to contain DNA, the majority of which was high mol wt. In conclusion, granulosa cells within the membrana granulosa die by apoptosis, with phagocytosis by a neighboring cell preceding any potential budding of the nucleus or cell itself. Granulosa cells near the antrum are sloughed off into the antrum, and their death has features more consistent with that of other cell types that undergo death as a result of terminal differentiation.

  12. Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1

    Science.gov (United States)

    Cells in the depth of the crypts in the bovine colon express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization 25 of cattle with human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used f...

  13. Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Muhammad Shahid

    2017-07-01

    Full Text Available Prototheca zopfii infections are becoming global concerns in humans and animals. Bovine protothecal mastitis is characterized by deteriorating milk quality and quantity, thus imparting huge economic losses to dairy industry. Previous published studies mostly focused on the prevalence and characterization of P. zopfii from mastitis. However, the ultrastructural pathomorphological changes associated with apoptosis in bovine mammary epithelial cells (bMECs are not studied yet. Therefore, in this study we aimed to evaluate the in vitro comparative apoptotic potential of P. zopfii genotype-I and -II on bMECs using flow cytometry, scanning electron microscopy (SEM, and transmission electron microscopy (TEM. The results showed fast growth rate and higher adhesion capability of genotype-II in bMECs as compared with genotype-I. The viability of bMECs infected with P. zopfii genotype-II was significantly decreased after 12 h (p < 0.05 and 24 h (p < 0.01 in comparison with control cells. Contrary, genotype-I couldn't show any significant effects on cell viability. Moreover, after infection of bMECs with genotype-II, the apoptosis increased significantly at 12 h (p < 0.05 and 24 h (p < 0.01 as compared with control group. Genotype-I couldn't display any significant effects on cell apoptosis. The host specificity of P. zopfii was also tested in mouse osteoblast cells, and the results suggest that genotype-I and -II could not cause any significant apoptosis in these cell lines. SEM interpreted the pathomorphological alterations in bMECs after infection. Adhesion of P. zopfii with cells and further disruption of cytomembrane validated the apoptosis caused by genotype-II under SEM. While genotype-1 couldn't cause any significant apoptosis in bMECs. Furthermore, genotype-II induced apoptotic manifested specific ultrastructure features, like cytoplasmic cavitation, swollen mitochondria, pyknosis, cytomembrane disruption, and appearance of apoptotic bodies under

  14. Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplatation embryos

    DEFF Research Database (Denmark)

    Jensen, Pernille L.; Beck, Hans Christian; Petersen, Tonny S.

    2014-01-01

    differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research......The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass...... describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early...

  15. Immunobiotics for the Bovine Host: Their Interaction with Intestinal Epithelial Cells and Their Effect on Antiviral Immunity

    Directory of Open Access Journals (Sweden)

    Julio Villena

    2018-03-01

    Full Text Available The scientific community has reported several cases of microbes that exhibit elevated rates of antibiotic resistance in different regions of the planet. Due to this emergence of antimicrobial resistant microorganisms, the use of antibiotics as promoters of livestock animals’ growth is being banned in most countries around the world. One of the challenges of agricultural immunology therefore is to find alternatives by modulating the immune system of animals in drug-independent safe food production systems. In this regard, in an effort to supplant antibiotics from bovine feeds, several alternatives were proposed including the use of immunomodulatory probiotics (immunobiotics. The purpose of this review is to provide an update of the status of the modulation of intestinal antiviral innate immunity of the bovine host by immunobiotics, and the beneficial impact of immunobiotics on viral infections, focused on intestinal epithelial cells (IECs. The results of our group, which demonstrate the capacity of immunobiotic strains to beneficially modulate Toll-like receptor 3-triggered immune responses in bovine IECs and improve the resistance to viral infections, are highlighted. This review provides comprehensive information on the innate immune response of bovine IECs against virus, which can be further investigated for the development of strategies aimed to improve defenses in the bovine host.

  16. Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures

    International Nuclear Information System (INIS)

    Bracken, W.M.; Sharma, R.P.; Bourcier, D.R.

    1984-01-01

    A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl 2 ) uptake and subcellular distribution had to cytotoxicity. Twenty-four-hour incubations with 0.05-50 μM HgCl 2 elicited a concentration-related cytotoxicity. Cellular accumulation of 203 Hg was also concentration-related, with 1.0 nmol/10 6 cells at the IC50. Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process. Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels. Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity. However, the subcellular distribution of Hg was not concentration-related. Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations. The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested. Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein. 31 references, 4 figures, 3 tables

  17. Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

    Science.gov (United States)

    Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

    2016-01-01

    The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

    International Nuclear Information System (INIS)

    Yonezawa, Tomo; Haga, Satoshi; Kobayashi, Yosuke; Katoh, Kazuo; Obara, Yoshiaki

    2008-01-01

    GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca 2+ concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival

  19. Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

    Science.gov (United States)

    Domingues, William Borges; da Silveira, Tony Leandro Rezende; Komninou, Eliza Rossi; Monte, Leonardo Garcia; Remião, Mariana Härter; Dellagostin, Odir Antônio; Corcini, Carine Dahl; Varela Junior, Antônio Sergio; Seixas, Fabiana Kömmling; Collares, Tiago; Campos, Vinicius Farias

    2017-08-01

    Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

  20. Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

    Science.gov (United States)

    Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

    2016-01-01

    Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

  1. Removal of glycosaminoglycans from bovine granulosa cells contributes to increased binding of hydrogen-3 heparin

    Energy Technology Data Exchange (ETDEWEB)

    Ax, R.L.; Stodd, C.M.; Boehm, S.K.; Bellin, M.E.

    1986-02-01

    Granulosa cells from small or large bovine follicles were pretreated with enzymes that hydrolyze various glycosaminoglycans, and binding of (/sup 3/H)-heparin to the granulosa was measured. Binding of (/sup 3/H) heparin increased significantly after enzymatic pretreatments with chondroitinase ABC and fungal hyaluronidase, and similar results were obtained with granulosa from small and large follicles. No changes in binding of (/sup 3/H) heparin were detected after hydrolyses with chondroitinase AC and heparinase in either follicle size. Heparitinase, which hydrolyzes heparan sulfate, led to a significant 50% increase in binding of (/sup 3/H) heparin to granulosa from large follicles but was without effect in small follicles. These results suggest that the lower binding of (/sup 3/H) heparin, which has been reported with follicular enlargement, may be due to heparan sulfate occupying or obstructing binding sites for heparin on granulosa from large follicles.

  2. Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

    Directory of Open Access Journals (Sweden)

    Fukuda Shin

    2009-11-01

    Full Text Available Abstract Background Theca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK. Methods Ovarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells. Results Akt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca

  3. Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells.

    Science.gov (United States)

    Klisch, Karl; Schraner, Elisabeth M; Boos, Alois

    2017-01-01

    Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate "BNC". Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering. © 2016 S. Karger AG, Basel.

  4. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-05-01

    Full Text Available Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH, apoptosis analysis by annexin V and propidium iodide (PI double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM and transmission electron microscope (TEM, mitochondrial transmembrane potential (ΔΨm assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05 release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01 and casepase-3 (p < 0.05 levels, significantly (p < 0.01 increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of

  5. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway.

    Science.gov (United States)

    Chen, Wei; Liu, Yongxia; Zhang, Limei; Gu, Xiaolong; Liu, Gang; Shahid, Muhammad; Gao, Jian; Ali, Tariq; Han, Bo

    2017-01-01

    Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz . 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly ( p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 ( p < 0.01) and casepase-3 ( p < 0.05) levels, significantly ( p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin

  6. CD154 costimulated ovine primary B cells, a cell culture system that supports productive infection by bovine leukemia virus.

    Science.gov (United States)

    Van den Broeke, A; Cleuter, Y; Beskorwayne, T; Kerkhofs, P; Szynal, M; Bagnis, C; Burny, A; Griebel, P

    2001-02-01

    Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.

  7. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Sanborn, B.B.; Schneider, A.S.

    1990-01-01

    Inositol trisphosphate (IP 3 ), a product of the phosphoinositide cycle, mobilizes intracellular Ca 2+ in many cell types. New evidence suggests that inositol tetrakisphosphate (IP 4 ), an IP 3 derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP 4 are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP 4 in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in [ 3 H]IP 4 and [ 3 H]IP 3 accumulation in chromaffin cells and this effect was completely blocked by atropine. [ 3 H]IP 4 accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP 3 and IP 4 hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP 4 and calcium homeostasis

  8. Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

    International Nuclear Information System (INIS)

    Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

    2010-01-01

    The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131 PRPR 134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

  9. Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

    International Nuclear Information System (INIS)

    Stepp, M.A.

    1986-01-01

    Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of [ 3 H]-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the α1 and α2 chains of type I and the α1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells

  10. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Víctor A. Solarte

    2015-01-01

    Full Text Available Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–254, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90% in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  11. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines.

    Science.gov (United States)

    Solarte, Víctor A; Rosas, Jaiver E; Rivera, Zuly J; Arango-Rodríguez, Martha L; García, Javier E; Vernot, Jean-Paul

    2015-01-01

    Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  12. Murine and bovine γδ T cells enhance innate immunity against Brucella abortus infections.

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    Jerod A Skyberg

    Full Text Available γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ(-/- mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα(-/-, and GMCSF(-/- mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ(-/- mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.

  13. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

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    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  14. Morphological changes in cultured bovine lymphoid cell lines associated with bovine viral diarrhea virus (BVDV) single and dual infections with bovine leukemia virus (BLV)

    Science.gov (United States)

    Currently, American Type Culture Collection (ATCC) makes available two cell lines derived from the same lymphoblast-like suspension cell that have been confirmed by next-generation sequencing and RT-PCR to have either a single contaminate of BVDV2a (CRL-8037) or dual contaminates of both BVDV and BL...

  15. Transition Metal Chelator Induces Progesterone Production in Mouse Cumulus-Oocyte Complexes and Corpora Lutea.

    Science.gov (United States)

    Tian, X; Anthony, K; Diaz, Francisco J

    2017-04-01

    Progesterone production is upregulated in granulosa cells (cumulus and mural) after the LH surge, but the intra-follicular mechanisms regulating this transition are not completely known. Recent findings show that the transition metal chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), impairs ovarian function. In this study, we provide evidence that chelating transition metals, including zinc, enhances progesterone production. The findings show that TPEN (transition metal chelator) increases abundance of Cyp11a1 and Star messenger RNA (mRNA) between 8- and 20-fold and progesterone production more than 3-fold in cultured cumulus-oocyte complexes (COC). Feeding a zinc-deficient diet for 10 days, but not 3 days, increased Star, Hsd3b, and prostaglandin F2 alpha receptor (Ptgfr) mRNA ~2.5-fold, suggesting that the effect of TPEN is through modulation of zinc availability. Progesterone from cumulus cells promotes oocyte developmental potential. Blocking progesterone production with epostane during maturation reduced subsequent blastocyst formation from 89 % in control to 18 % in epostane-treated complexes, but supplementation with progesterone restored blastocyst developmental potential to 94 %. Feeding a zinc-deficient diet for 5 days before ovulation did not affect the number of CL, STAR protein, or serum progesterone. However, incubating luteal tissue with TPEN increased abundance of Star, Hsd3b, and Ptgfr mRNA 2-3-fold and increased progesterone production 3-fold. TPEN is known to abolish SMAD2/3 signaling in cumulus cells. However, treatment of COC with the SMAD2/3 phosphorylation inhibitor, SB421542, did not by itself induce steroidogenic transcripts but did potentiate EGF-induced Star mRNA expression. Collectively, the results show that depletion of transition metals with TPEN acutely enhances progesterone biosynthesis in COC and luteal tissue.

  16. Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model.

    Science.gov (United States)

    Schmutzer, Michael; Aszodi, Attila

    2017-04-01

    The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

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    Gabriella Mamede Andrade

    Full Text Available The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells and cumulus-oocyte complexes (COCs to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

  18. Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells

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    Pirson Chris

    2012-06-01

    Full Text Available Abstract Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM and trehalose monomycolate (TMM, the apolar phthiocerol dimycocersates (PDIMs, triacyl glycerol (TAG, pentacyl trehalose (PAT, phenolic glycolipid (PGL, and mono-mycolyl glycerol (MMG. Polar lipids identified included glucose monomycolate (GMM, diphosphatidyl glycerol (DPG, phenylethanolamine (PE and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs. These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.

  19. Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

    Science.gov (United States)

    Touzard, Eve; Reinaud, Pierrette; Dubois, Olivier; Guyader-Joly, Catherine; Humblot, Patrice; Ponsart, Claire; Charpigny, Gilles

    2013-10-01

    Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

  20. Simulation of solar radiative transfer in cumulus clouds

    Energy Technology Data Exchange (ETDEWEB)

    Zuev, V.E.; Titov, G.A. [Institute of Atmospheric Optics, Tomsk (Russian Federation)

    1996-04-01

    This work presents a 3-D model of radiative transfer which is used to study the relationship between the spatial distribution of cumulus clouds and fluxes (albedo and transmittance) of visible solar radiation.

  1. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    Science.gov (United States)

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  2. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

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    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  3. Bovine Peripheral Blood Mononuclear Cells Are More Sensitive to Deoxynivalenol Than Those Derived from Poultry and Swine

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    Barbara Novak

    2018-04-01

    Full Text Available Deoxynivalenol (DON is one of the most prevalent mycotoxins, contaminating cereals and cereal-derived products. Its derivative deepoxy-deoxynivalenol (DOM-1 is produced by certain bacteria, which either occur naturally or are supplemented in feed additive. DON-induced impairments in protein synthesis are particularly problematic for highly proliferating immune cells. This study provides the first comparison of the effects of DON and DOM-1 on the concanavalin A-induced proliferation of porcine, chicken, and bovine peripheral blood mononuclear cells (PBMCs. Therefore, isolated PBMCs were treated with DON (0.01–3.37 µM and DOM-1 (1.39–357 µM separately, and proliferation was measured using a bromodeoxyuridine (BrdU assay. Although pigs are considered highly sensitive to DON, the present study revealed a substantially higher sensitivity of bovine (IC50 = 0.314 µM PBMCs compared to chicken (IC50 = 0.691 µM and porcine (IC50 = 0.693 µM PBMCs. Analyses on the proliferation of bovine T-cell subsets showed that all major subsets, namely, CD4+, CD8β+, and γδ T cells, were affected to a similar extent. In contrast, DOM-1 did not affect bovine PBMCs, but reduced the proliferation of chicken and porcine PBMCs at the highest tested concentration (357 µM. Results confirm the necessity of feed additives containing DON-to-DOM-1-transforming bacteria and highlights species-specific differences in the DON sensitivity of immune cells.

  4. Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

    Science.gov (United States)

    Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

    1995-06-01

    We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

  5. Making the switch: alternatives to foetal bovine serum for adipose-derived stromal cell expansion

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    Carla Dessels

    2016-10-01

    Full Text Available Adipose-derived stromal cells (ASCs are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing procedures (GMPs and are safe for use in humans. The majority of clinical trials in which ASCs are expanded make use of fetal bovine serum (FBS. While FBS is used traditionally in the research setting for in vitro expansion, it does carry the risk of xenoimmunization and zoonotic transmission when used for expanding cells destined for therapeutic purposes. In order to ensure a GMP quality product for cellular therapy, in vitro expansion of ASCs has been undertaken using xeno-free (XF, chemically-defined, and human blood-derived alternatives. These investigations usually include the criteria proposed by the International Society of Cellular Therapy (ISCT and International Fat Applied Technology Society (IFATS. The majority of studies use these criteria to compare plastic-adherence, morphology, the immunophenotype and the trilineage differentiation of ASCs under the different medium supplemented conditions. Based on these studies, all of the alternatives to FBS seem to be suitable replacements; however, each has its own advantages and drawbacks. Very few studies have investigated the effects of the supplements on the immunomodulation of ASCs; the transcriptome, proteome and secretome; and the ultimate effects in appropriate animal models. The selection of medium supplementation will depend on the downstream application of the ASCs and their efficacy and safety in preclinical studies.

  6. Radiolocalization of bovine lymphosarcoma cells in athymic mice, using a monoclonal antibody against tumor-associated antigens

    International Nuclear Information System (INIS)

    Aida, Y.; Ochiai, K.; Ito, K.; Onuma, M.; Fujimori, F.; Fujimoto, Y.; Izawa, H.

    1987-01-01

    Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125 I. The 125 I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography

  7. Advanced glycation end-products (AGEs acutely impair Ca2+ signalling in bovine aortic endothelial cells

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    Nadim eNaser

    2013-03-01

    Full Text Available Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca2+] and Ca2+ signalling, the aim of this study was to examine the acute effects of AGEs on Ca2+ signalling in bovine aortic endothelial cells (BAEC. Ca2+ signalling was studied using the fluorescent indicator dye Fura2-AM. AGEs were generated by incubating bovine serum albumin with 0 - 250 mM glucose or glucose-6-phosphate for 0 to 120 days at 37ºC. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML as assayed using both GC-MS and HPLC. In Ca2+-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated the increase in intracellular [Ca2+] caused by ATP (100 µM. In the absence of extracellular Ca2+, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca2+] and attenuated subsequent intracellular Ca2+ release caused by ATP, thapsigargin (0.1 µM and ionomycin (3 µM, but AGEs did not affect extracellular Ca2+ entry induced by the re-addition of Ca2+ to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 µM and NAD(PH oxidase inhibitors apocynin (500 µM and diphenyleneiodonium (DPI, 1 µM abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 µM. In summary, AGEs caused an acute depletion of Ca2+ from the intracellular store in BAECs, such that the Ca2+ signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of ROS from NAD(PH oxidase and possible activation of the IP3 receptor.

  8. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

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    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  9. Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells

    OpenAIRE

    Hillreiner, Maria;Flinspach, Claudia;Pfaffl, Michael W.;Kliem, Heike

    2017-01-01

    Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC) challenged with the mastitis pathogen Escherichia coli (E. coli). Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from m...

  10. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Fitzpatrick, D.R.; Zamb, T.; Parker, M.D.; van Drunen Littel-van den Hurk, S.; Babiuk, L.A.; Lawman, M.J.P.

    1988-01-01

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK - cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  11. Microphysical imprint of entrainment in warm cumulus

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    Jennifer D. Small

    2013-07-01

    Full Text Available We analyse the cloud microphysical response to entrainment mixing in warm cumulus clouds observed from the CIRPAS Twin Otter during the GoMACCS field campaign near Houston, Texas, in summer 2006. Cloud drop size distributions and cloud liquid water contents from the Artium Flight phase-Doppler interferometer in conjunction with meteorological observations are used to investigate the degree to which inhomogeneous versus homogeneous mixing is preferred as a function of height above cloud base, distance from cloud edge and aerosol concentration. Using four complete days of data with 101 cloud penetrations (minimum 300 m in length, we find that inhomogeneous mixing primarily explains liquid water variability in these clouds. Furthermore, we show that there is a tendency for mixing to be more homogeneous towards the cloud top, which we attribute to the combination of increased turbulent kinetic energy and cloud drop size with altitude which together cause the Damköhler number to increase by a factor of between 10 and 30 from cloud base to cloud top. We also find that cloud edges appear to be air from cloud centres that have been diluted solely through inhomogeneous mixing. Theory predicts the potential for aerosol to affect mixing type via changes in drop size over the range of aerosol concentrations experienced (moderately polluted rural sites to highly polluted urban sites. However, the observations, while consistent with this hypothesis, do not show a statistically significant effect of aerosol on mixing type.

  12. Progesterone Receptor Membrane Component 1 (PGRMC1 in cell division: its role in bovine granulosa cells mitosis

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    Laura Terzaghi

    2015-07-01

    Full Text Available The present studies were aimed to assess Progesterone Receptor Membrane Component-1 (PGRMC1 role in regulating bovine granulosa cells (bGC mitosis. First, we performed immunofluorescence studies on in vitro cultured bGC collected from antral follicles, which showed that PGRMC1 localizes to the spindle apparatus in mitotic cells. Then, to evaluate PGRMC1 effect on cell proliferation we silenced its expression with RNA interference technique (RNAi. Quantitative RT-PCR and immunoblotting confirmed down-regulation of PGRMC1 expression, when compared to CTRL-RNAi treated bGC (p<0.05. After 72h of culture, PGRMC1 silencing determined a lower growth rate (p<0.05 and a higher percentage of cells arrested at G2/M phase as assessed by flowcytometry (p<0.05. Accordingly, live imaging studies revealed more aberrant mitosis and a delayed M-phase in PGRMC1-RNAi treated cells compared to CTRL-RNAi group (p<0.05. These data confirmed that PGRMC1 is directly involved in bGC mitosis and ongoing preliminary studies are aimed to elucidate its putative mechanisms of action. Since PGRMC1 is a membrane protein, we hypothesize its possible involvement in vesicular trafficking and endocytosis, which is in turn an important process to assure proper cell division. To assess this hypothesis, we have preliminarily conducted immunofluorescence and in situ proximity ligation assay experiments that showed PGRMC1 co-localization and direct interaction with clathrin. This is important since clathrin is an essential protein for both endosomes formation, and cell division acting directly on the spindle apparatus. Thus our studies set the stage for analysis aimed to further characterize PGRMC1’s mechanism of action in mitotic cell.

  13. Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.

    Science.gov (United States)

    Ziegler, Anja; Everett, Helen; Hamza, Eman; Garbani, Mattia; Gerber, Vinzenz; Marti, Eliane; Steinbach, Falko

    2016-11-15

    Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies upon a combination of GM-CSF and IL-4 added to cell culture medium which is supplemented with FBS. The aim of this study was to substitute FBS with heterologous horse serum. For this purpose, equine monocyte-derived dendritic cells (eqMoDC) were generated in the presence of horse serum or FBS and analysed for the effect on morphology, phenotype and immunological properties. Changes in the expression of phenotypic markers (CD14, CD86, CD206) were assessed during dendritic cell maturation by flow cytometry. To obtain a more complete picture of the eqMoDC differentiation and assess possible differences between FBS- and horse serum-driven cultures, a transcriptomic microarray analysis was performed. Lastly, immature eqMoDC were primed with a primary antigen (ovalbumin) or a recall antigen (tetanus toxoid) and, after maturation, were co-cultured with freshly isolated autologous CD5 + T lymphocytes to assess their T cell stimulatory capacity. The microarray analysis demonstrated that eqMoDC generated with horse serum were indistinguishable from those generated with FBS. However, eqMoDC incubated with horse serum-supplemented medium exhibited a more characteristic dendritic cell morphology during differentiation from monocytes. A significant increase in cell viability was also observed in eqMoDC cultured with horse serum. Furthermore, eqMoDC generated in the presence of horse serum

  14. Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2009-01-01

    Summary Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 C/min in a low-temperature (80 C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; 20 C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type. ?? 2009 Cambridge University Press.

  15. SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

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    He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

    2017-01-01

    Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

  16. Low oxygen level increases proliferation and metabolic changes in bovine granulosa cells.

    Science.gov (United States)

    Shiratsuki, Shogo; Hara, Tomotaka; Munakata, Yasuhisa; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-12-05

    The present study addresses molecular backgrounds underlying low oxygen induced metabolic changes and 1.2-fold change in bovine granulosa cell (GCs) proliferation. RNA-seq revealed that low oxygen (5%) upregulated genes associated with HIF-1 and glycolysis and downregulated genes associated with mitochondrial respiration than that in high oxygen level (21%). Low oxygen level induced high glycolytic activity and low mitochondrial function and biogenesis. Low oxygen level enhanced GC proliferation with high expression levels of HIF-1, VEGF, AKT, mTOR, and S6RP, whereas addition of anti-VEGF antibody decreased cellular proliferation with low phosphorylated AKT and mTOR expression levels. Low oxygen level reduced SIRT1, whereas activation of SIRT1 by resveratrol increased mitochondrial replication and decreased cellular proliferation with reduction of phosphorylated mTOR. These results suggest that low oxygen level stimulates the HIF1-VEGF-AKT-mTOR pathway and up-regulates glycolysis, which contributes to GC proliferation, and downregulation of SIRT1 contributes to hypoxia-associated reduction of mitochondria and cellular proliferation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes.

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    Ming-Hong Sun

    Full Text Available As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs and cumulus-enclosed oocytes (CEOs. We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.

  18. Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

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    RAÚL VINET

    2009-01-01

    Full Text Available Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca2+ dependent exocitosis of vasoactive compounds. Using the Ca2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs respond to acetylcholine (ACh with a cytosolic Ca2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23. The increase in cytosolic Ca2+ induced by 10µM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 µM of hexamethonium. On the other hand, the increase in cytosolic Ca2+ could be depressed by nifedipine (0.01 -100 µM or withdrawal of extracellular Ca2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca2+ channels, influx of external Ca2+ and liberation of vasoactive compounds.

  19. Hyaluronan Protects Bovine Articular Chondrocytes against Cell Death Induced by Bupivacaine under Supraphysiologic Temperatures

    Science.gov (United States)

    Liu, Sen; Zhang, Qing-Song; Hester, William; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

    2013-01-01

    Background Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination these two deleterious factors could further impair cell viability. Hypothesis Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design Controlled laboratory study. Methods Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for one hour, and then exposed to bupivacaine for one hour at room temperature. Cell viability was assessed at three time points: immediately after treatment, six hours later, and twenty-four hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison to the phosphate-buffered saline control group; and, (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at six and twenty-four hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusions Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance This study provides a rationale to perform pre-clinical and clinical studies to

  20. Bovine neonatal pancytopenia--comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK).

    Science.gov (United States)

    Euler, Kerstin N; Hauck, Stefanie M; Ueffing, Marius; Deeg, Cornelia A

    2013-01-23

    Bovine neonatal pancytopenia (BNP) is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV) was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney) cells, the cell line used for production of the associated vaccine. By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

  1. Bovine neonatal pancytopenia - Comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK

    Directory of Open Access Journals (Sweden)

    Euler Kerstin N

    2013-01-01

    Full Text Available Abstract Background Bovine neonatal pancytopenia (BNP is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney cells, the cell line used for production of the associated vaccine. Results By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. Conclusions The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

  2. Fundamental aspects of bovine oocyte maturation : the role of estradiol, VIP and GHRH

    NARCIS (Netherlands)

    Beker van Woudenberg, A.R.C.L. (Anna Rita Costa Lage)

    2004-01-01

    Chapter 1 presents an overview on the aspects of oocyte maturation. Growth hormone (GH), released from the pituitary by the stimulus of GHRH, increases cumulus expansion and improves cytoplasmic maturation in bovine oocytes. GHRH is also expressed in extraneural tissues suggesting that GHRH also

  3. Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

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    F.D. Oliveira

    2015-12-01

    Full Text Available In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1 of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

  4. Primary bovine skeletal muscle cells enters apoptosis rapidly via the intrinsic pathway when available oxygen is removed.

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    Sissel Beate Rønning

    Full Text Available Muscle cells undergo changes post-mortem during the process of converting muscle into meat, and this complex process is far from revealed. Recent reports have suggested programmed cell death (apoptosis to be important in the very early period of converting muscle into meat. The dynamic balance that occurs between anti-apoptotic members, such as Bcl-2, and pro-apoptotic members (Bid, Bim helps determine whether the cell initiates apoptosis. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to investigate if apoptosis is induced when oxygen is removed from the growth medium. Primary bovine muscle cells were differentiated to form myotubes, and anoxia was induced for 6h. The anoxic conditions significantly increased (P<0.05 the relative gene expression of anti- and pro-apoptotic markers (Aif, Bcl-2, Bid and Bim, and the PARK7 (P<0.05 and Grp75 (Hsp70 protein expressions were transiently increased. The anoxic conditions also led to a loss of mitochondrial membrane potential, which is an early apoptotic event, as well as cytochrome c release from the mitochondria. Finally, reorganization and degradation of cytoskeletal filaments occurred. These results suggest that muscle cells enters apoptosis via the intrinsic pathway rapidly when available oxygen in the muscle diminishes post-mortem.

  5. Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

    Science.gov (United States)

    Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

    2009-09-01

    Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

  6. INFLUÊNCIA DA TEMPERATURA DE FECUNDAÇÃO IN VITRO E DAS CÉLULAS DO Cumulus oophorus SOBRE A TAXA DE POLISPERMIA E DESENVOLVIMENTO EMBRIONÁRIO

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    Melissa Savoia Castilho Cunha

    2010-06-01

    Full Text Available Effects of in vitro fertilization (IVF temperature andcumulus oophorus cells removal after IVF or 12 h of embryoculture (IVC on polyspermy and embryo development rates wereevaluated in swine. Oocytes and spermatozoa were incubated at37 or 38.5ºC during IVF procedure. Cumulus oophorus cells wereremoved from 50% of zygotes of each group 8 hours after IVF andall zygotes were cultured with NCSU23 media. Polyspermy rateswere assessed after 12 hours of IVC, when cumulus oophorus cellswere removed from the rest of zygotes. In a second experiment,embryos remained in culture for the evaluation of embryodevelopment. No effects of IVF temperature or cumulus oophoruscells removal were observed after 12 hours of IVC on polyspermyand embryo development (p<0.05. In conclusion, IVF temperatureand the presence of cumulus oophorus cells after IVF do notinterfere on polyspermy and embryo development rates.

  7. Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

    Science.gov (United States)

    Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

    2016-10-01

    To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

  8. Oxygen sensitivity of potassium- and angiotensin II-stimulated aldosterone release by bovine adrenal cells.

    Science.gov (United States)

    Brickner, R C; Raff, H

    1991-04-01

    Angiotensin II (AII) and extracellular K+, acting through different intracellular mechanisms, stimulate aldosterone release in a synergistic fashion. We have previously shown that decreases in oxygen (O2) within the physiological range inhibit AII, cyclic AMP (cAMP) and ACTH-stimulated aldosterone release. The present experiment evaluated the effect of various concentrations of O2 on K+-stimulated aldosterone release in the presence and absence of AII. Dispersed bovine adrenal glomerulosa cells were incubated with different concentrations of K+ (0.9-5.4 mmol/l) without and with AII (10 nmol/l) under different concentrations of O2 (0, 5 or 50%); 21% O2 (pO2 = 19.9 +/- 0.5 kPa,n = 9) was used as reference control for comparison. In all cases, increases in K+ stimulated aldosterone release, an effect augmented by AII. Under 0% O2 (pO2 = 8.1 +/- 0.3 kPa, n = 3) and 5% O2 (pO2 = 12.8 +/- 0.5 kPa, n = 3), aldosterone release stimulated by K+ or K+/AII was significantly inhibited compared with that under 21% O2. Conversely, under 50% O2 (pO2 = 36.3 +/- 2.5 kPa, n = 3), aldosterone release stimulated by K+ or K+/AII was significantly augmented. Cortisol secretion was not significantly affected by 5% or 50% O2 but was significantly decreased under 0% O2. The effect of O2 on K+/AII stimulation of aldosterone release, as well as previous experiments with cAMP, progesterone and ACTH, suggest a final common post-receptor oxygen-sensitive component of the aldosterone synthetic pathway. It is suggested that one or more enzymes in the aldosterone synthetic pathway is/are exquisitely sensitive to small changes in O2 within the physiological range.

  9. RESEARCHES REGARDING THE INFLUENCE OF THE NUMBER OF CUMULAR CELLS LAYER OVER THE OOCYTE MATURATION EFFICIENCY

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    V. CARABĂ

    2009-05-01

    Full Text Available During the experiments we have carried out with imature oocyte collected from the ovarian follicles, wefound a variety of oocyte-cumulus complexes. We got the following experiment in order to understand therole of cumular cells on the achievement of the cytoplasma and oocyte nucleus maturation. We select theoocyte-cumulus complexes collected both from cows and sows according to the number of cumular celllayers and we watched their development to the blastocyst stade. Thus, we achieved three groups of COC(oocyte-cumulus complexes.One group was made of oocyte without cumular cells, the second group had a layer of cumular cells andthe third group had many layers of cumular cells. we performed an incubation of all these types of COCin TCM-199 enriched with 20% of bovine fetal serum. Because only 1,2 oocyte of the ones who lack thecumular cells layer had maturation signs during cultivation in the thermostat versus 55 and 115,respectively, of the ones that had many cellular layers, presents a solid evidence that cumular cells areindispensable for the maturation and even to the fecundation process. The cumular cells perform adecisive role on the cytoplasma and oocyte nucleus maturation process.

  10. β-Hydroxybutyrate Facilitates Fatty Acids Synthesis Mediated by Sterol Regulatory Element-Binding Protein1 in Bovine Mammary Epithelial Cells

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    Min Zhang

    2015-11-01

    Full Text Available Background/Aims: In dairy cows, β-hydroxybutyrate (BHBA is utilized as precursors of de novo synthesized fatty acids in mammary gland. Ketotic cows are characterized by excessive negative energy balance (NEB, which can further increase the blood BHBA concentration. Sterol regulatory element-binding protein1 (SREBP1 and cell death-inducing DNA fragmentation factor-alpha-like effector α (Cidea play crucial roles in lipid synthesis. Therefore, we hypothesized that BHBA could stimulate SREBP1/Cidea pathway to increase milk fat synthesis in bovine mammary epithelial cells. Methods: Bovine mammary epithelial cells were treated with different concentrations of BHBA and transfected with adenovirus to silence SREBP1 expression. The effects of BHBA on the lipid synthesis in bovine mammary epithelial cells were investigated. Results: The results showed that BHBA could significantly increase the expression of SREBP1, fatty acid synthase (FAS, acetyl-CoA carboxylase α (ACC-α, Cidea and diacylglycerol transferase-1 (DGAT-1, as well as the triglycerides (TG content in bovine mammary epithelial cells. BHBA treatment also increased the transfer of mature SREBP1 to nucleus compared with control group. However, SREBP1 silencing could significantly down-regulate the overexpression of FAS, ACC-α, Cidea and DGAT-1, as well as TG content induced by BHBA. Conclusion: The present data indicate that BHBA can significantly increase TG secretion mediated by SREBP1 in bovine mammary epithelial cells.

  11. Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

    Science.gov (United States)

    Zhang, Jin-You; Wu, Yi; Zhao, Shuan; Liu, Zhen-Xing; Zeng, Shen-Ming; Zhang, Gui-Xue

    2015-09-15

    Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells.

    Science.gov (United States)

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Lu, Zengjun; Bao, Huifang; Bai, Xingwen; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2016-07-15

    The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Hotelli Cumulus Mikkelin majoitusasiakkaiden tyytyväisyys

    OpenAIRE

    Kinnunen, Jenni Ljuba

    2016-01-01

    Tämän opinnäytetyön tavoitteena on tutkia majoitusasiakkaiden tyytyväisyyttä hotelli Cumulus Mikkelissä keskittyen pääosin hotellin vastaanottoon, hotellihuoneeseen, Huviretki-ravintolaan ja aamiaiseen. Opinnäytetyön toimeksiantaja on hotelli Cumulus Mikkeli. Tässä opinnäytetyössä käytetään kvalitatiivista tutkimusmenetelmää ja tutkimusaineisto kerättiin haastattelemalla hotellin majoitusasiakkaita yksitellen kyselylomakkeen kanssa. Haastateltujen asiakkaiden määrä oli 20, joista 10 ...

  14. Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells.

    Science.gov (United States)

    Li, Ran; Dudemaine, Pier-Luc; Zhao, Xin; Lei, Chuzhao; Ibeagha-Awemu, Eveline Mengwi

    2016-01-01

    alternative non-invasive source of RNA in assessing miRNA activities in bovine mammary gland. Predicted target genes (1802) of 14 highly expressed miRNAs in milk fractions were enriched in fundamental cellular functions, infection, organ and tissue development. Furthermore, some miRNAs were highly enriched (FDR whey (3), cells (11) and mammary gland tissue (14) suggesting specific regulatory functions in the various fractions. In conclusion, we have obtained a comprehensive miRNA profile of the different milk fractions using high throughput sequencing. Our comparative analysis showed that miRNAs from milk fat accurately portrayed the miRNome of mammary gland tissue. Functional annotation of the top expressed miRNAs in milk confirmed their critical regulatory roles in mammary gland functions and potentially to milk recipients.

  15. Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

    Science.gov (United States)

    Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

    1999-08-01

    We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

  16. IFN-τ Mediated Control of Bovine Major Histocompatibility Complex Class I Expression and Function via the Regulation of bta-miR-148b/152 in Bovine Endometrial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Haichong Wu

    2018-02-01

    Full Text Available IFN-τ, a type I interferon produced by the trophoblasts of ruminants, has various important immune functions, including effects on the expression of major histocompatibility complex (MHC class I (MHC-I. A previous study has reported that IFN-τ promotes the expression of MHC-I molecules on endometrial cells. However, the immunological mechanisms by which IFN-τ regulates MHC-I molecules remain unknown. Here, we investigated which microRNA (miRNAs may be involved in the regulation of MHC-I molecule expression and function in bovine endometrial epithelial cells (bEECs. By using TargetScan 6.2 and http://www.microRNA.org, two miRNAs were suggested to target the 3′UTR of the bovine MHC-I heavy chain: bta-miR-148b and bta-miR-152. Dual luciferase reporter and miRNA mimic/inhibitor assays suggested that bta-miR-148b/152 were negatively correlated with bovine MHC-I heavy chain genes. The function of the MHC-I heavy chain was then investigated using qRT-PCR, ELISA, western blotting, immunofluorescence, and RNA interference assays in primary bEECs and an endometrial epithelial cell line (BEND. The results demonstrated that bta-miR-148b/152 could promote TLR4-triggered inflammatory responses by targeting the bovine MHC-I heavy chain, and the MHC-I molecule negatively regulated TLR4-induced inflammatory reactions may through the Fps-SHP-2 pathway. Our discovery offers novel insight into negative regulation of the TLR4 pathway and elucidates the mechanism by which bovine MHC-I molecules control congenital inflammatory reactions.

  17. Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

    Directory of Open Access Journals (Sweden)

    Li CongJun

    2006-09-01

    Full Text Available Abstract Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Discovery Rate (FDR = 0 % were identified. The majority of these genes were repressed by butyrate and associated with cell cycle control. The expression levels of 30 selected genes identified by the microarray were confirmed using real-time PCR. The results from real-time PCR positively correlated (R = 0.867 with the results from the microarray. Conclusion This study presented the genes related to multiple signal pathways such as cell cycle control and apoptosis. The profound changes in gene expression elucidate the molecular basis for the pleiotropic effects of butyrate on biological processes. These findings enable better recognition of the full range of beneficial roles butyrate may play during cattle energy metabolism, cell growth and proliferation, and possibly in fighting gastrointestinal pathogens.

  18. Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load.

    Science.gov (United States)

    Nagasawa, Yuya; Kiku, Yoshio; Sugawara, Kazue; Tanabe, Fuyuko; Hayashi, Tomohito

    2018-01-01

    The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary-infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3-21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin-positive cells in the milk from the infused udders (r = 0.734, P mastitis udders caused by S. aureus may contribute to reduced milk yield. © 2017 Japanese Society of Animal Science.

  19. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    Science.gov (United States)

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  20. Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

    1998-03-01

    Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

  1. Pericardial Parietal Mesothelial Cells: Source of the Angiotensin-Converting-Enzyme of the Bovine Pericardial Fluid

    Directory of Open Access Journals (Sweden)

    Ilsione Ribeiro de Sousa Filho

    Full Text Available Abstract Background: Angiotensin II (Ang II, the primary effector hormone of the renin-angiotensin system (RAS, acts systemically or locally, being produced by the action of angiotensin-converting-enzyme (ACE on angiotensin I. Although several tissue RASs, such as cardiac RAS, have been described, little is known about the presence of an RAS in the pericardial fluid and its possible sources. Locally produced Ang II has paracrine and autocrine effects, inducing left ventricular hypertrophy, fibrosis, heart failure and cardiac dysfunction. Because of the difficulties inherent in human pericardial fluid collection, appropriate experimental models are useful to obtain data regarding the characteristics of the pericardial fluid and surrounding tissues. Objectives: To evidence the presence of constituents of the Ang II production paths in bovine pericardial fluid and parietal pericardium. Methods: Albumin-free crude extracts of bovine pericardial fluid, immunoprecipitated with anti-ACE antibody, were submitted to electrophoresis (SDS-PAGE and gels stained with coomassie blue. Duplicates of gels were probed with anti-ACE antibody. In the pericardial membranes, ACE was detected by use of immunofluorescence. Results: Immunodetection on nitrocellulose membranes showed a 146-KDa ACE isoform in the bovine pericardial fluid. On the pericardial membrane sections, ACE was immunolocalized in the mesothelial layer. Conclusions: The ACE isoform in the bovine pericardial fluid and parietal pericardium should account at least partially for the production of Ang II in the pericardial space, and should be considered when assessing the cardiac RAS.

  2. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

    NARCIS (Netherlands)

    Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

    2013-01-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

  3. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

    Science.gov (United States)

    Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

    2010-02-18

    The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

  4. Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

    Science.gov (United States)

    Alves-Barroco, Cinthia; Roma-Rodrigues, Catarina; Raposo, Luís R; Brás, Catarina; Diniz, Mário; Caço, João; Costa, Pedro M; Santos-Sanches, Ilda; Fernandes, Alexandra R

    2018-03-25

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  5. Impact of cloud microphysics and cumulus parameterization on ...

    Indian Academy of Sciences (India)

    2007-10-09

    Oct 9, 2007 ... Bangladesh. Weather Research and Forecast (WRF–ARW version) modelling system with six dif- .... tem intensified rapidly into a land depression over southern part of ... Impact of cloud microphysics and cumulus parameterization on heavy rainfall. 261 .... tent and temperature and is represented as a sum.

  6. Laboratory simulations of cumulus cloud flows explain the entrainment anomaly

    Science.gov (United States)

    Narasimha, Roddam; Diwan, Sourabh S.; Subrahmanyam, Duvvuri; Sreenivas, K. R.; Bhat, G. S.

    2010-11-01

    In the present laboratory experiments, cumulus cloud flows are simulated by starting plumes and jets subjected to off-source heat addition in amounts that are dynamically similar to latent heat release due to condensation in real clouds. The setup permits incorporation of features like atmospheric inversion layers and the active control of off-source heat addition. Herein we report, for the first time, simulation of five different cumulus cloud types (and many shapes), including three genera and three species (WMO Atlas 1987), which show striking resemblance to real clouds. It is known that the rate of entrainment in cumulus cloud flows is much less than that in classical plumes - the main reason for the failure of early entrainment models. Some of the previous studies on steady-state jets and plumes (done in a similar setup) have attributed this anomaly to the disruption of the large-scale turbulent structures upon the addition of off-source heat. We present estimates of entrainment coefficients from these measurements which show a qualitatively consistent variation with height. We propose that this explains the observed entrainment anomaly in cumulus clouds; further experiments are planned to address this question in the context of starting jets and plumes.

  7. Anti-inflammatory effects of conjugated linoleic acid isomers and essential fatty acids in bovine mammary epithelial cells.

    Science.gov (United States)

    Dipasquale, D; Basiricò, L; Morera, P; Primi, R; Tröscher, A; Bernabucci, U

    2018-01-09

    Fatty acids are important modulators of inflammatory responses, in particular, n-3 and n-6 essential fatty acids and CLA have received particular attention for their ability to modulate inflammation. The objectives of this study were to compare the effects of CLA and essential fatty acids on the expression of pro and anti- inflammatory cytokines and their protective efficacy against inflammatory status in mammary gland by an in vitro model based on bovine mammary epithelial cells (BME-UV1). Bovine mammary epithelial cells were treated with complete medium containing either 50 µM of cis-9, trans-11 CLA (c9,t11 CLA) or trans-10, cis-12 CLA (t10,c12 CLA) or (α)-linolenic acid (aLnA) or (γ)-linolenic acid (gLnA) or linoleic acid (LA). After 48 h by fatty acids administration the cells were treated for 3 h with 20 µM of lipopolysaccharide (LPS) to induce inflammatory stimulus. Reactive oxygen species (ROS) production after treatments was assessed to verify and to compare the potential protection of different fatty acids against LPS-induced oxidative stress. The messenger RNA abundance of bovine pro and anti-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukine-10 (IL-10)) and peroxisome proliferator receptor-α/γ (PPARγ/α) were determined in BME-UV1 by real-time PCR. The results showed that cells treated with fatty acids and LPS increased ROS production compared with control cells. Among treatments, cells treated with c9,t11 CLA and t10,c12 CLA isomers revealed significant lower levels of ROS production compared with other fatty acids. All fatty acids reduced the gene expression of pro- and anti-inflammatory cytokines. Among fatty acids, t10,c12 CLA, LA and gLnA showed an homogeneous reduction of the three pro-inflammatory cytokines and this may correspond to more balanced and efficient physiological activity and may trigger a better protective effect. The PPARγ gene expression was

  8. Cumulus parameterizations in chemical transport models

    Science.gov (United States)

    Mahowald, Natalie M.; Rasch, Philip J.; Prinn, Ronald G.

    1995-12-01

    Global three-dimensional chemical transport models (CTMs) are valuable tools for studying processes controlling the distribution of trace constituents in the atmosphere. A major uncertainty in these models is the subgrid-scale parametrization of transport by cumulus convection. This study seeks to define the range of behavior of moist convective schemes and point toward more reliable formulations for inclusion in chemical transport models. The emphasis is on deriving convective transport from meteorological data sets (such as those from the forecast centers) which do not routinely include convective mass fluxes. Seven moist convective parameterizations are compared in a column model to examine the sensitivity of the vertical profile of trace gases to the parameterization used in a global chemical transport model. The moist convective schemes examined are the Emanuel scheme [Emanuel, 1991], the Feichter-Crutzen scheme [Feichter and Crutzen, 1990], the inverse thermodynamic scheme (described in this paper), two versions of a scheme suggested by Hack [Hack, 1994], and two versions of a scheme suggested by Tiedtke (one following the formulation used in the ECMWF (European Centre for Medium-Range Weather Forecasting) and ECHAM3 (European Centre and Hamburg Max-Planck-Institut) models [Tiedtke, 1989], and one formulated as in the TM2 (Transport Model-2) model (M. Heimann, personal communication, 1992). These convective schemes vary in the closure used to derive the mass fluxes, as well as the cloud model formulation, giving a broad range of results. In addition, two boundary layer schemes are compared: a state-of-the-art nonlocal boundary layer scheme [Holtslag and Boville, 1993] and a simple adiabatic mixing scheme described in this paper. Three tests are used to compare the moist convective schemes against observations. Although the tests conducted here cannot conclusively show that one parameterization is better than the others, the tests are a good measure of the

  9. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

    NARCIS (Netherlands)

    Naaijkens, B.A.; Niessen, H.W.M.; Prins, H.J.; Krijnen, P.A.J.; Kokhuis, T.J.A.; de Jong, N.; van Hinsbergh, V.W.M.; Kamp, O.; Helder, M.N.; Musters, R.J.P.; van Dijk, A.; Juffermans, L.J.M.

    2012-01-01

    Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

  10. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

    NARCIS (Netherlands)

    B. Naaijkens (Benno); H.W.M. Niessen (Hans ); H.-J. Prins (H.); P.A.J. Krijnen (Paul); T.J.A. Kokhuis (Tom); N. de Jong (Nico); V.W.M. van Hinsbergh (Victor); O. Kamp (Otto); K. Helder MScN (Onno); R.J.P. Musters (René); A. van Dijk (Annemieke); L.J.M. Juffermans (Lynda)

    2012-01-01

    textabstractAdipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

  11. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induced autophagy in a bovine kidney cell line

    International Nuclear Information System (INIS)

    Fiorito, Filomena; Ciarcia, Roberto; Granato, Giovanna Elvira; Marfe, Gabriella; Iovane, Valentina; Florio, Salvatore; De Martino, Luisa; Pagnini, Ugo

    2011-01-01

    Highlights: ► TCDD induces cell proliferation in MDBK cells. ► TCDD provokes morphological cell death in MDBK cells. ► TCDD induces autophagy in MDBK cells. ► TCDD induces a down-regulation of telomerase activity, bTERT, c-Myc in MDBK cells. ► TCDD induces an up-regulation of p53 and p21 protein levels in MDBK cells. -- Abstract: The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK) cells where no signs of apoptosis were observed, but herein, analysis of MDBK cell morphology, in a large number of exposed cells, revealed some alterations, as expanded cytoplasm, an increase of intercellular spaces and many pyknotic nuclei. Hence, the aim of the current study was to elucidate the influences of dioxin on cell proliferation and cell death. We found that dioxin increased proliferation, as well as, activated cell death with autophagy, as we detected by increased amount of LC3-II, an autophagosome marker. Furthermore, formation of acidic vesicular organelles was observed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. These results were accompanied by down-regulation of telomerase activity, bTERT and c-Myc. Key tumor-suppressor protein p53 and expression of cell cycle inhibitor p21Waf1/Cip1 were activated after TCDD exposure. These changes occurred with activation of ATM phosphorylation in the presence of a decrease in Mdm2 protein levels. Taken together, these results support the idea that TCDD in MDBK cells, may exert its action, in part, by enhancing cell proliferation, but also by modulating the incidence of induced cell death with autophagy.

  12. Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

    Science.gov (United States)

    Blomström, Anne-Lie; Gu, Quan; Barry, Gerald; Wilkie, Gavin; Skelton, Jessica K; Baird, Margaret; McFarlane, Melanie; Schnettler, Esther; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

    2015-04-19

    Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

  13. TGF-β1 Induces EMT in Bovine Mammary Epithelial Cells Through the TGFβ1/Smad Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Qing Chen

    2017-08-01

    Full Text Available Background/Aims: Transforming growth factor-β1 (TGF-β1 plays a crucial role in chronic inflammation in various tissues, and is related to inflammation-caused organ fibrogenesis associated with the epithelial-mesenchymal transition (EMT and the deposition of the extracellular matrix (ECM. However, the effect of TGF-β1 on bovine mammary epithelial cells (BMECs with mastitis, and its mechanism, remain unknown. Methods: We analyzed the level of TGF-β1 in inflamed mammary tissues and cells using western blotting. BMECs were treated with TGF-β1, and EMT-related gene and protein expression changes were evaluated using quantitative real-time polymerase chain reaction (qPCR, western blotting, and immunofluorescence. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor, and analyzed EMT-related protein expression by western blotting. In addition, we injected TGF-β1 into mice mammary glands to investigate whether it can cause mammary fibrosis in vivo. Results: The TGF-β1 level was up-regulated in mammary tissues with mastitis and in inducible inflammatory BMECs. TGF-β1 treatment activated the TGF/ Smad signaling pathway in BMECs during their transition to the EMT phenotype, as indicated by morphological changes from a cobblestone-like shape to a spindle-like one. TGF-β1 treatment also up-regulated the expression of α-smooth muscle actin, vimentin, and collagen I, albumin, and down-regulated the expression of E-cadherin both in mRNA level and protein level. Furthermore, TGF-β1 enhanced the gene expressions of MMP2, MMP7, and fibronectin in BMECs. TGF-β1 injection induced mice mammary infection and fibrosis. Conclusion: These findings suggested that aberrant up-regulation of TGF-β1 in bovine mastitic mammary glands might play an important role in bovine mammary fibrosis caused by unresolved inflammation.

  14. Early Detection of Rapidly Developing Cumulus Area using HIMAWARI-8

    Science.gov (United States)

    Yamada, Y.; Kadosaki, G.

    2017-12-01

    In recent years, many disasters have been occured by influence of meteorological change in Japan. So, it becomes more important to inform rapid weather change caused by cumulus which brings concentrated heavy rain/hail, wind gust, lightning in a short period. These severe events should inclease in the future by global warming. Therefore we are developping the alert system for Rapidly Developing Cumulus Area (RDCA) detection using Japanese new satellite. At July 2015, Japan Meteorological Agency started operation of new geostationary meteorological satellite "Himawari-8". This satellite has optical imager named Advanced Himawari Imager (AHI). It can observe Japan area every 2.5 minutes. The frequently infrared image with high resolution (2km) is the key of our alert system. We took some special functions in the algorithm of this system. One of the points is cloud location which shifts to north from true location around Japan by viewing angle from the satellite above the equator. We moved clouds to the correct position using geometric correction method according to its height and latitude. This algorithm also follows a movement of cloud every 2.5 minutes during several observations. It derives the information about degree of the development of cumulus. The prototype system gives the alert before 30 to 60 minutes in advance to the first lightning in typical cumulus case. However, we understand that there are some difficult cases to alert. For example, winter low cloud over the Japan Sea which brings a winter lightning, and tornado (although it is not cumulus). Now, we are adjusting some parameters of the algorithm. In the near future, our algorithm will be used in weather information delivery service to the customer.

  15. Influence of oxygen and hydrogen treated graphene on cell adhesion in the presence or absence of fetal bovine serum

    International Nuclear Information System (INIS)

    Verdanova, Martina; Broz, Antonin; Kalbac, Martin; Kalbacova, Marie

    2012-01-01

    The influence of differently treated graphene on human osteoblasts after 2 h of incubation with regard to the presence/absence of fetal bovine serum (FBS) was investigated. Cell adhesion plays an important role in further cell fate and it is influenced by cell surrounding. It was found that treatment of graphene (by hydrogen or oxygen) does not play role in number of cells which adhere to substrate after 2 h of incubation. However, it is important for cell size - cells are larger on the hydrogen treated graphene than on the oxygen treated graphene. The presence of FBS is crucial for a type of interaction between cells and their substrate - in the presence of FBS, interactions are mediated by specific proteins and thus formation of focal adhesions (FAs) can occur. However, in the absence of FBS, a contact is carried out by non-specific bonds without FAs formation. It was observed that cells on graphene samples without FBS have star-like shape and larger area in contrast to cells adhering with FBS which have round shape and are smaller. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  16. Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

    Science.gov (United States)

    Kim, Eun Young; Park, Min Jee; Park, Hyo Young; Noh, Eun Ji; Noh, Eun Hyung; Park, Kyoung Sik; Lee, Jun Beom; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2012-08-01

    In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

  17. Porcine cumulus and mural granulosa cells secrete cumulus expansion enabling factor (CEEF)

    Czech Academy of Sciences Publication Activity Database

    Procházka, Radek; Nagyová, Eva; Motlík, Jan

    1996-01-01

    Roč. 39, special issue (1996), s. 86 ISSN 0003-9438. [Satelite Symposium of the Central European Conference on Animal Reproduction /1./. Kühlungsborn, 22.09.1996-23.09.1996] R&D Projects: GA ČR GA505/93/2010; GA AV ČR IAA745401 Grant - others:GZ 45379/1-IV/3a/94 Source of funding: AT

  18. Influence of the fetal bovine serum proteins on the growth of human osteoblast cells on graphene

    Czech Academy of Sciences Publication Activity Database

    Kalbáčová, M.; Brož, A.; Kalbáč, Martin

    100A, č. 11 (2012), s. 3001-3007 ISSN 1549-3296 R&D Projects: GA AV ČR IAA400400911; GA AV ČR KAN200100801; GA ČR GAP204/10/1677; GA ČR(CZ) GAP208/12/1062; GA MŠk ME09060 Institutional support: RVO:61388955 Keywords : human osteoblast * graphene * fetal bovine serum Subject RIV: CG - Electrochemistry Impact factor: 2.834, year: 2012

  19. Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

    Science.gov (United States)

    Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

    2014-01-01

    Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

  20. Proteomic Analysis of Bovine Nucleolus

    Institute of Scientific and Technical Information of China (English)

    Amrutlal K.Patel; Doug Olson; Suresh K. Tikoo

    2010-01-01

    Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

  1. Bovine lactoferrin and lactoferricin exert antitumor activities on human colorectal cancer cells (HT-29) by activating various signaling pathways.

    Science.gov (United States)

    Jiang, Rulan; Lönnerdal, Bo

    2017-02-01

    Lactoferrin (Lf) is an iron-binding glycoprotein that is present at high concentrations in milk. Bovine lactoferricin (LfcinB) is a peptide fragment generated by pepsin proteolysis of bovine lactoferrin (bLf). LfcinB consists of amino acid residues 17-41 proximal to the N-terminus of bLf and a disulfide bond between residues 19 and 36, forming a loop. Both bLf and LfcinB have been demonstrated to have antitumor activities. Colorectal cancer is the second most common cause of cancer death in developed countries. We hypothesized that bLf and LfcinB exert antitumor activities on colon cancer cells (HT-29) by triggering various signaling pathways. bLf and LfcinB significantly induced apoptosis in HT-29 cells but not in normal human intestinal epithelial cells, as revealed by the ApoTox-Glo Triplex Assay. The LIVE/DEAD cell viability assay showed that both bLf and LfcinB reduced the viability of HT-29 cells. Transcriptome analysis indicated that bLf, cyclic LfcinB, and linear LfcinB exerted antitumor activities by differentially activating diverse signaling pathways, including p53, apoptosis, and angiopoietin signaling. Immunoblotting results confirmed that both bLf and LfcinBs increased expression of caspase-8, p53, and p21, critical proteins in tumor suppression. These results provide valuable information regarding bLf and LfcinB for potential clinical applications in colon cancer therapy.

  2. Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage

    Directory of Open Access Journals (Sweden)

    Anja Baufeld

    2018-02-01

    Full Text Available Abstract Background The LH-induced folliculo-luteal transformation is connected with alterations of the gene expression profile in cells of the granulosa layer. It has been described that hypoxic conditions occur during luteinization, thus favoring the formation of L-lactate within the follicle. Despite being a product of anaerobic respiration, L-lactate has been shown to act as a signaling molecule affecting gene expression in neuronal cells. During the present study, we tested the hypothesis that L-lactate may influence differentiation of follicular granulosa cells (GC. Methods In a bovine granulosa cell culture model effects of L- and D-lactate, of increased glucose concentrations and of the lactate transport inhibitor UK5099 were analyzed. Steroid hormone production was analyzed by RIA and the abundance of key transcripts was determined by quantitative real-time RT-PCR. Results L-lactate decreased the production of estradiol and significantly affected selected genes of the folliculo-luteal transition as well as genes of the lactate metabolism. CYP19A1, FSHR, LHCGR were down-regulated, whereas RGS2, VNN2, PTX3, LDHA and lactate transporters were up-regulated. These effects could be partly or completely reversed by pre-treatment of the cells with UK5099. The non-metabolized enantiomer D-lactate had even more pronounced effects on gene expression, whereas increased glucose concentrations did not affect transcript abundance. Conclusions In summary, our data suggest that L-lactate specifically alters physiological and molecular characteristics of GC. These effects critically depend on L-lactate uptake, but are not triggered by increased energy supply. Further, we could show that L-lactate has a positive feedback on the lactate metabolism. Therefore, we hypothesize that L-lactate acts as a signaling molecule in bovine and possibly other monovular species supporting differentiation during the folliculo-luteal transformation.

  3. Microplasma jet treatment of bovine serum albumin coatings for controlling enzyme and cell attachmenttype="fn" rid="FN1">

    Science.gov (United States)

    Szili, Endre J.; Becker, Stefanie; Short, Robert D.; Al-Bataineh, Sameer A.

    2017-08-01

    We investigated a new approach to control protein and cell attachment inside 96-well polystyrene plates. The wells were first coated with bovine serum albumin (BSA) to inhibit cell and protein attachment. The BSA-coated wells were then treated with a helium microplasma jet for increasing times that resulted in gradual removal of BSA from the surface. It was found that the amount of enzyme and cell attachment could be controlled in the wells where BSA was only partially removed by the microplasma jet. In addition to the surface coverage of BSA, the new surface chemistry induced by the microplasma jet treatment also had an important role in the control of enzyme and cell attachment. In summary, microplasma jet treatment of BSA-coated polystyrene wells is a simple and effective method for controlling enzyme and cell attachment. This might find use for high-throughput screening of new cell culture platforms where control over the level protein, enzyme or cell adherence is needed in order to maintain a specific cell function.

  4. Molecular mechanisms of insulin-like growth factor 1 promoted synthesis and retention of hyaluronic acid in porcine oocyte-cumulus complexes

    Czech Academy of Sciences Publication Activity Database

    Němcová, Lucie; Nagyová, Eva; Petlach, Michal; Tománek, M.; Procházka, Radek

    2007-01-01

    Roč. 76, - (2007), s. 1016-1024 ISSN 0006-3363 R&D Projects: GA ČR GA523/04/0574 Institutional research plan: CEZ:AV0Z50450515 Keywords : cumulus cells * expansion * follicle-stimulating hormone Subject RIV: ED - Physiology Impact factor: 3.670, year: 2007

  5. Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides

    Czech Academy of Sciences Publication Activity Database

    Blaha, Milan; Němcová, Lucie; Vodičková Kepková, Kateřina; Vodička, P.; Procházka, Radek

    2015-01-01

    Roč. 13, č. 113 (2015) ISSN 1477-7827 R&D Projects: GA ČR GAP502/11/0593; GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : FSH * growth factors * cumulus cell Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.147, year: 2015

  6. Toxicological effects of fumonisin B1 alone and in combination with other fusariotoxins on bovine granulosa cells.

    Science.gov (United States)

    Albonico, Marco; Schütz, Luis F; Caloni, Francesca; Cortinovis, Cristina; Spicer, Leon J

    2016-08-01

    There is now overwhelming evidence of global contamination of commodities with Fusarium mycotoxins. Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON), α-zearalenol (α-ZEA) and β-zearalenol (β-ZEA). The aim of this study was to determine if FB1, alone and combined with DON or α-ZEA or β-ZEA, can affect cell proliferation and steroid production of bovine granulosa cells (GC). A species-specific model with bovine granulosa cells (GC) was used to study the potential endocrine disruptor effects of FB1 alone and in co-exposure. In the presence of β-ZEA (30 ng/mL), FB1 at 30 ng/mL showed a stimulatory effect on GC numbers. Insulin-like growth factor-1 (IGF1)-stimulated cell proliferation was decreased after exposure to β-ZEA alone at 5.0 μg/mL and FB1 with α-ZEA and β-ZEA at the same concentration. Regarding steroid production, FB1 at 30 ng/mL and 100 ng/mL amplified the inhibitory effect of β-ZEA (30 ng/mL) on estradiol (E2) production, while FB1 alone increased (P < 0.05) IGF1-induced E2 production. α-ZEA alone decreased (P < 0.05) E2 production, whereas β-ZEA alone and in combination with FB1 decreased (P < 0.05) E2 production. These studies indicate for the first time that the Fusarium mycotoxin FB1 along with other mycotoxins can affect GC proliferation and steroid production, which ultimately could influence reproductive function in cattle. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

    Science.gov (United States)

    Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

    2018-04-13

    Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P lipofection', may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

  8. Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid.

    Science.gov (United States)

    Yoneyama, Tohru; Miyata, Keita; Chikai, Tomoyuki; Mikami, Akifumi; Suzuki, Tomonori; Hasegawa, Kimiko; Ikeda, Toshihiko; Watanabe, Toshihiro; Ohyama, Tohru; Niwa, Koichi

    2008-12-01

    Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

  9. Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Maria Hillreiner

    Full Text Available Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC challenged with the mastitis pathogen Escherichia coli (E. coli. Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes.

  10. Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

    Science.gov (United States)

    Hillreiner, Maria; Flinspach, Claudia; Pfaffl, Michael W; Kliem, Heike

    2016-01-01

    Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC) challenged with the mastitis pathogen Escherichia coli (E. coli). Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes.

  11. Transbilayer transport of a propyltrimethylammonium derivative of diphenylhexatriene (TMAP-DPH) in bovine blood platelets and adrenal chromaffin cells.

    Science.gov (United States)

    Kitagawa, Shuji; Tachikawa, Eiichi; Kashimoto, Takashi

    2002-12-01

    The membrane fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium (TMAP-DPH) has an additional three-carbon spacer between the fluorophore and the trimethylammonium substituent of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). As a basic study to clarify the transport mechanism of amphiphilic quaternary ammoniums, we observed the characteristics of the transbilayer transport of TMAP-DPH in bovine blood platelets and bovine adrenal chromaffin cells using the albumin extraction method. We compared these inward transport rates with those of TMA-DPH. TMAP-DPH crossed into the cytoplasmic layers of the membranes more slowly than TMA-DPH after rapid binding to the outer halves of the plasma membranes. The transport rate markedly depended on temperature. Time to reach the half-maximal incorporated amount of TMAP-DPH increased threefold accompanied by an increase in the concentration from 0.2 to 1.5 microM. The transport was stimulated significantly by various types of membrane perturbations such as modification of sulfhydryl-groups by N-ethylmaleimide and benzyl alcohol-induced increase in the fluidity of the lipid bilayer. The saturation phenomenon suggested the presence of the regulatory process in the transbilayer transport of TMAP-DPH.

  12. The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.

    Science.gov (United States)

    Kim, Eun Young; Lee, Jun Beom; Park, Hyo Young; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2011-06-01

    Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; Pculture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.

  13. Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos.

    Science.gov (United States)

    Sutton-McDowall, Melanie L; Feil, Deanne; Robker, Rebecca L; Thompson, Jeremy G; Dunning, Kylie R

    2012-05-01

    Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  14. Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin.

    Science.gov (United States)

    Furlong, Suzanne J; Ridgway, Neale D; Hoskin, David W

    2008-03-01

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

  15. Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

    Directory of Open Access Journals (Sweden)

    Edda de Rizzo

    1984-04-01

    Full Text Available Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE. As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80 than the latter (1:80 to 1:160. Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

  16. Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton, via the non-structural protein 3Cpro

    DEFF Research Database (Denmark)

    Armer, Hannah; Moffat, Katy; Wileman, Thomas

    2008-01-01

    Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus r....... In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect -tubulin distribution, and the microtubule network remained relatively unaffected....

  17. Identification of candidate vaccine antigens of bovine hemoparasites Theileria parva and Babesia bovis by use of helper T cell clones.

    Science.gov (United States)

    Brown, W C; Zhao, S; Logan, K S; Grab, D J; Rice-Ficht, A C

    1995-03-01

    Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of

  18. Methyl mercury uptake across bovine brain capillary endothelial cells in vitro: The role of amino acids

    International Nuclear Information System (INIS)

    Aschner, M.; Clarkson, T.W.

    1989-01-01

    Previous studies in the rat in vivo have demonstrated that co-injection of methyl mercury (MeHg) with L-cysteine into the common carotid artery enhances brain Hg levels folowing a single capillary pass through the CNS vasculature. In order to elucidate the relationship between MeHg transport and the neutral amino acid transport carrier system, regulatory aspects of MeHg transport across the bovine blood-brain barrier were investigated in isolated brain microvessel preparations. Following 1 hour co-incubations of 203 Hg-MeHgCl with 0.1 mM L-cysteine at 37 deg. C, 203 Hg uptake by suspended microvessels was significantly increased (P 203 Hg was abolished by co-incubations of microvessels with 0.1 mM L-cysteine-L-methionine, or 0.1 mM L-cysteine plus AT-125 (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazolacetic acid), an irreversible inhibitor of gamma-glutamyl-transpeptidase. One hr co-incubations of bovine capilaries with 203 Hg-MeHgCl and 0.1 mM D-cysteine at 37 deg. C or 0.1 mM L-cysteine at 0 deg. did not increase rat of 203 Hg uptake compared with controls. These results indicate that L-cysteine enhances the rate of capillary MeHg uptake. The accumulation of 203 Hg in the bovine microvessels appears to be a carrier-mediated process. It is inhibited by L-methionin, a competitive substrate for neutral amino acid transport, and by AT-125. Capillary uptake of 203 Hg is stereospecific to the L-enantiomorph of cystine, suggesting selective uptake of MeHg across the blood-brain barrier. The data emphasize the relationship between the L-enantiomorph neutral amino acid carrier system and MeHg transport across the capillaries. (author)

  19. Laboratory Studies of Anomalous Entrainment in Cumulus Cloud Flows

    Science.gov (United States)

    Diwan, Sourabh S.; Narasimha, Roddam; Bhat, G. S.; Sreenivas, K. R.

    2011-12-01

    Entrainment in cumulus clouds has been a subject of investigation for the last sixty years, and continues to be a central issue in current research. The development of a laboratory facility that can simulate cumulus cloud evolution enables us to shed light on the problem. The apparatus for the purpose is based on a physical model of cloud flow as a plume with off-source diabatic heating that is dynamically similar to the effect of latent-heat release in natural clouds. We present a critical review of the experimental data so far obtained in such facilities on the variation of the entrainment coefficient in steady diabatic jets and plumes. Although there are some unexplained differences among different data sets, the dominant trend of the results compares favourably with recent numerical simulations on steady-state deep convection, and helps explain certain puzzles in the fluid dynamics of clouds.

  20. Laboratory Studies of Anomalous Entrainment in Cumulus Cloud Flows

    International Nuclear Information System (INIS)

    Diwan, Sourabh S; Narasimha, Roddam; Sreenivas, K R; Bhat, G S

    2011-01-01

    Entrainment in cumulus clouds has been a subject of investigation for the last sixty years, and continues to be a central issue in current research. The development of a laboratory facility that can simulate cumulus cloud evolution enables us to shed light on the problem. The apparatus for the purpose is based on a physical model of cloud flow as a plume with off-source diabatic heating that is dynamically similar to the effect of latent-heat release in natural clouds. We present a critical review of the experimental data so far obtained in such facilities on the variation of the entrainment coefficient in steady diabatic jets and plumes. Although there are some unexplained differences among different data sets, the dominant trend of the results compares favourably with recent numerical simulations on steady-state deep convection, and helps explain certain puzzles in the fluid dynamics of clouds.

  1. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    Science.gov (United States)

    Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

    2016-01-01

    Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003

  2. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    Directory of Open Access Journals (Sweden)

    Erika Evangelina Coronado-Cerda

    2016-01-01

    Full Text Available Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE or IMMUNEPOTENT CRP® (ICRP is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM, cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients.

  3. The MTT assays of bovine retinal pericytes and human microvascular endothelial cells on DLC and Si-DLC-coated TCPS.

    Science.gov (United States)

    Okpalugo, T I T; McKenna, E; Magee, A C; McLaughlin, J; Brown, N M D

    2004-11-01

    MTT (Tetrazolium)-assay suggests that diamond-like carbon (DLC) and silicon-doped DLC (Si-DLC) films obtained under appropriate deposition parameters are not toxic to bovine retinal pericytes, and human microvascular endothelial cells (HMEC). The observed frequency distributions of the optical density (OD) values indicative of cell viability are near Gaussian-normal distribution. One-way ANOVA indicates that at 0.05 levels the population means are not significantly different for the coated and control samples. The observed OD values depend on the cell line (cell growth/metabolic rate), possibly cell cycle stage, the deposition parameters-bias voltage, ion energy, pressure, argon precleaning, and the dopant. For colored thin films like DLC with room temperature photoconductivity and photoelectric effects, it is important to account for the OD contribution from the coating itself. MTT assay, not surprisingly, seems not to be highly sensitive to interfacial cellular interaction resulting from the change in the film's nanostructure, because the tetrazolium metabolism is mainly intracellular and not interfacial. The thin films were synthesized by 13.56 MHz RF-PECVD using argon and acetylene as source gases, with tetramethylsilane (TMS) vapor introduced for silicon doping. This study could be relevant to biomedical application of the films in the eye, peri-vascular, vascular compartments, and for cell-tissue engineering. (c) 2004 Wiley Periodicals, Inc.

  4. The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

    Science.gov (United States)

    Randau, Thomas M; Schildberg, Frank A; Alini, Mauro; Wimmer, Matthias D; Haddouti, El-Mustapha; Gravius, Sascha; Ito, Keita; Stoddart, Martin J

    2013-01-01

    The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal

  5. Transcriptomal profiling of bovine ovarian granulosa and theca interna cells in primary culture in comparison with their in vivo counterparts.

    Directory of Open Access Journals (Sweden)

    Nicholas Hatzirodos

    Full Text Available In vitro culture of ovarian granulosa cells and theca cells has been very important for our understanding of their function and regulation. One of the most eagerly sought attributes of cell culture is the use of chemically-defined conditions. However, even under such in vitro conditions cell behaviour could differ from the in vivo situation because of differences in oxygen tension, nutrients, adhesion matrix and other factors. To examine this further we compared the transcriptomes of both granulosa cells and cells from the theca interna that were cultured in what are arguably the best in vitro conditions for maintaining the 'follicular' phenotypes of both tissue types, as displayed by their respective freshly-isolated counterparts. The array data analysed are from recently published data and use the same sizes of bovine follicles (small antral 3-6 mm and the same Affymetrix arrays. We conducted analysis using Partek, Ingenuity Pathway Analysis and GOEAST. Principal Component Analysis (PCA and hierarchical clustering clearly separated the in vivo from the in vitro groups for both cells types and transcriptomes were more homogeneous upon culture. In both cell cultures behaviours associated with cell adhesion, migration and interaction with matrix or substrate were more abundant. However, the pathways involved generally differed between the two cell types. With the thecal cultures a gene expression signature of an immune response was more abundant, probably by leukocytes amongst the cells cultured from the theca interna. These results indicate differences between in vivo and in vitro that should be considered when interpreting in vitro data.

  6. Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

    Science.gov (United States)

    van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

    2017-11-25

    We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

  7. The anti-epileptic drug valproic acid (VPA inhibits steroidogenesis in bovine theca and granulosa cells in vitro.

    Directory of Open Access Journals (Sweden)

    Claire Glister

    Full Text Available Valproic acid (VPA is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC and granulosa (GC cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P99% decrease; P<0.0001 with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05. VPA only reduced TC progesterone secretion induced by the highest (luteinizing LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001 by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001. The potent histone deacetylase (HDAC inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

  8. Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

    Science.gov (United States)

    Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

    2013-12-01

    Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Impact of in vitro treatments of physiological levels of estradiol and progesterone observed in pregnancy on bovine monocyte-derived dendritic cell differentiation and maturation

    NARCIS (Netherlands)

    Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    The specific factors which regulate differentiation and maturation of dendritic cells in bovine pregnancy remain unclear. We evaluated the influence of physiologically relevant in vitro treatments of progesterone (PG) and estradiol (E2) observed in late pregnancy on the differentiation and

  10. Impact of in vitro treatments of physiological levels of estradiol and progesterone observed in pregnancy on bovine monocyte-derived dendritic cell differentiation and maturation

    NARCIS (Netherlands)

    Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    The specific factors which regulate differentiation and maturation of dendritic cells in bovine pregnancy remain unclear. We evaluated the influence of physiologically relevant in vitro treatments of progesterone (PG) and estradiol (E2) observed in late pregnancy on the differentiation and

  11. Characterization of bovine gamma delta T cells phenotype during post-natal development and following Mycobacterium bovis vaccination or virulent infection

    Science.gov (United States)

    Bovine tuberculosis caused by Mycobacterium bovis is a globally significant veterinary health problem. Gamma delta T cells are known to participate in the immune control of mycobacterial infections. Data in human and non-human primates suggest that mycobacterial infection regulates memory/effector p...

  12. Fluctuations in a quasi-stationary shallow cumulus cloud ensemble

    Directory of Open Access Journals (Sweden)

    M. Sakradzija

    2015-01-01

    Full Text Available We propose an approach to stochastic parameterisation of shallow cumulus clouds to represent the convective variability and its dependence on the model resolution. To collect information about the individual cloud lifecycles and the cloud ensemble as a whole, we employ a large eddy simulation (LES model and a cloud tracking algorithm, followed by conditional sampling of clouds at the cloud-base level. In the case of a shallow cumulus ensemble, the cloud-base mass flux distribution is bimodal, due to the different shallow cloud subtypes, active and passive clouds. Each distribution mode can be approximated using a Weibull distribution, which is a generalisation of exponential distribution by accounting for the change in distribution shape due to the diversity of cloud lifecycles. The exponential distribution of cloud mass flux previously suggested for deep convection parameterisation is a special case of the Weibull distribution, which opens a way towards unification of the statistical convective ensemble formalism of shallow and deep cumulus clouds. Based on the empirical and theoretical findings, a stochastic model has been developed to simulate a shallow convective cloud ensemble. It is formulated as a compound random process, with the number of convective elements drawn from a Poisson distribution, and the cloud mass flux sampled from a mixed Weibull distribution. Convective memory is accounted for through the explicit cloud lifecycles, making the model formulation consistent with the choice of the Weibull cloud mass flux distribution function. The memory of individual shallow clouds is required to capture the correct convective variability. The resulting distribution of the subgrid convective states in the considered shallow cumulus case is scale-adaptive – the smaller the grid size, the broader the distribution.

  13. The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures.

    Science.gov (United States)

    McCann, K B; Lee, A; Wan, J; Roginski, H; Coventry, M J

    2003-01-01

    To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.

  14. Continuous growth of cloud droplets in cumulus cloud

    International Nuclear Information System (INIS)

    Gotoh, Toshiyuki; Suehiro, Tamotsu; Saito, Izumi

    2016-01-01

    A new method to seamlessly simulate the continuous growth of droplets advected by turbulent flow inside a cumulus cloud was developed from first principle. A cubic box ascending with a mean updraft inside a cumulus cloud was introduced and the updraft velocity was self-consistently determined in such a way that the mean turbulent velocity within the box vanished. All the degrees of freedom of the cloud droplets and turbulence fields were numerically integrated. The box ascended quickly inside the cumulus cloud due to the updraft and the mean radius of the droplets grew from 10 to 24 μ m for about 10 min. The turbulent flow tended to slow down the time evolutions of the updraft velocity, the box altitude and the mean cloud droplet radius. The size distribution of the cloud droplets in the updraft case was narrower than in the absence of the updraft. It was also found that the wavenumeber spectra of the variances of the temperature and water vapor mixing ratio were nearly constant in the low wavenumber range. The future development of the new method was argued. (paper)

  15. Entrainment in Laboratory Simulations of Cumulus Cloud Flows

    Science.gov (United States)

    Narasimha, R.; Diwan, S.; Subrahmanyam, D.; Sreenivas, K. R.; Bhat, G. S.

    2010-12-01

    A variety of cumulus cloud flows, including congestus (both shallow bubble and tall tower types), mediocris and fractus have been generated in a water tank by simulating the release of latent heat in real clouds. The simulation is achieved through ohmic heating, injected volumetrically into the flow by applying suitable voltages between diametral cross-sections of starting jets and plumes of electrically conducting fluid (acidified water). Dynamical similarity between atmospheric and laboratory cloud flows is achieved by duplicating values of an appropriate non-dimensional heat release number. Velocity measurements, made by laser instrumentation, show that the Taylor entrainment coefficient generally increases just above the level of commencement of heat injection (corresponding to condensation level in the real cloud). Subsequently the coefficient reaches a maximum before declining to the very low values that characterize tall cumulus towers. The experiments also simulate the protected core of real clouds. Cumulus Congestus : Atmospheric cloud (left), simulated laboratory cloud (right). Panels below show respectively total heat injected and vertical profile of heating in the laboratory cloud.

  16. The Inhibition Effect of Cell DNA Oxidative Damage and LDL Oxidation by Bovine Colostrums

    Directory of Open Access Journals (Sweden)

    Chih-Wei Chen

    2016-10-01

    Full Text Available In the present study, we investigated the effect of bovine colostrums on inhibition of DNA oxidative damage and low density lipoprotein (LDL oxidation in vitro. Results showed that whey and skimmed milk exhibited not only higher inhibitory activities of oxidative damage of deoxyribose but also an inhibitory effect on the breakdown of supercoiled DNA into open circular DNA and linear DNA. The quantities of 8-OH-2′-dG formed under whey, caseins and skimmed milk treatment were 0.24, 0.24 and 1.24 μg/mL, respectively. The quantity of malondialdehyde formed through LDL oxidation induced by copprous ion was significantly decreased as colostrums protein solutions were added, in which whey and caseins led to a more significant decrease than skimmed milk. The formation of conjugated dienes could be inhibited by treatment with colostrums protein solutions. Whey exhibited the longest lag time of conjugated dienes formation among the colostrums proteins. The lag time of the whey was 2.33 times that of the control. From the results of foregoing, the bovine colostrums protein has potential value in the inhibition of DNA oxidation damage and LDL oxidation.

  17. Ketamine inhibits 45Ca influx and catecholamine secretion by inhibiting 22Na influx in cultured bovine adrenal medullary cells

    International Nuclear Information System (INIS)

    Takara, Hiroshi; Wada, Akihiko; Arita, Masahide; Izumi, Futoshi; Sumikawa, Koji

    1986-01-01

    The effects of ketamine, an intravenous anesthetic, on 22 Na influx, 45 Ca influx and catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Ketamine inhibited carbachol-induced 45 Ca influx and catecholamine secretion in a concentration-dependent manner with a similar potency. Ketamine also reduced veratridine-induced 45 Ca influx and catecholamine secretion. The influx of 22 Na caused by carbachol or by veratridine was suppressed by ketamine with a concentration-inhibition curve similar to that of 45 Ca influx and catecholamine secretion. Inhibition by ketamine of the carbachol-induced influx of 22 Na, 45 Ca and secretion of catecholamines was not reversed by the increased concentrations of carbachol. These observations indicate that ketamine, at clinical concentrations, can inhibit nicotinic receptor-associated ionic channels and that the inhibition of Na influx via the receptor-associated ionic channels is responsible for the inhibition of carbachol-induced Ca influx and catecholamine secretion. (Auth.)

  18. Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: mechanism of action and reversible inhibition by brevenal.

    Science.gov (United States)

    Nguyen-Huu, Truong D; Mattei, César; Wen, Peter J; Bourdelais, Andrea J; Lewis, Richard J; Benoit, Evelyne; Baden, Daniel G; Molgó, Jordi; Meunier, Frédéric A

    2010-10-01

    Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera. Copyright 2009 Elsevier Ltd. All rights reserved.

  19. The affection of APA microcapsulation on catecholamine and leucine-enkephalin secretion from the bovine adrenal medullary chromaffin cells

    International Nuclear Information System (INIS)

    Guo Shuilong; Cui Xin; Luo Yun; Xue Yilong

    2002-01-01

    The affection of alginate-polylysine-alginate (APA) microcapsulation on catecholamine (CA) and leucine-enkephalin (L-EK) secretion from bovine adrenal medullary chromaffin cells was analysed. Encapsulating BCCs with the APA microcapsulation, the secretion of CA and L-EK in encapsulated BCCs was detected by high-performance liquid chromatography-electrochemical assay and radioimmunoassay, respectively. There is little difference between the encapsulated BCCs and the non-encapsulated BCCs in the secretion of epinephrine (E) and noradrenaline (NE) but the secretion of dopamine (DA) and L-EK in several points decline. The studies indicated that APA microcapsulation of BCCs didn't affect the secretion of E and NE, but did the secretion DA and L-EK in forepart

  20. Immunomodulatory Effects of Amblyomma variegatum Saliva on Bovine Cells: Characterization of Cellular Responses and Identification of Molecular Determinants

    Directory of Open Access Journals (Sweden)

    Valérie Rodrigues

    2018-01-01

    Full Text Available The tropical bont tick, Amblyomma variegatum, is a tick species of veterinary importance and is considered as one of major pest of ruminants in Africa and in the Caribbean. It causes direct skin lesions, transmits heartwater, and reactivates bovine dermatophilosis. Tick saliva is reported to affect overall host responses through immunomodulatory and anti-inflammatory molecules, among other bioactive molecules. The general objective of this study was to better understand the role of saliva in interaction between the Amblyomma tick and the host using cellular biology approaches and proteomics, and to discuss its impact on disease transmission and/or activation. We first focused on the immuno-modulating effects of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMC and monocyte-derived macrophages in vitro. We analyzed its immuno-suppressive properties by measuring the effect of saliva on PBMC proliferation, and observed a significant decrease in ConA-stimulated PBMC lymphoproliferation. We then studied the effect of saliva on bovine macrophages using flow cytometry to analyze the expression of MHC-II and co-stimulation molecules (CD40, CD80, and CD86 and by measuring the production of nitric oxide (NO and pro- or anti-inflammatory cytokines. We observed a significant decrease in the expression of MHC-II, CD40, and CD80 molecules, associated with decreased levels of IL-12-p40 and TNF-α and increased level of IL-10, which could explain the saliva-induced modulation of NO. To elucidate these immunomodulatory effects, crude saliva proteins were analyzed using proteomics with an Orbitrap Elite mass spectrometer. Among the 336 proteins identified in A. variegatum saliva, we evidenced bioactive molecules exhibiting anti-inflammatory, immuno-modulatory, and anti-oxidant properties (e.g., serpins, phospholipases A2, heme lipoprotein. We also characterized an intriguing ubiquitination complex that could be involved in

  1. IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.

    Science.gov (United States)

    Gajewska, Małgorzata; Motyl, Tomasz

    2004-10-01

    TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway.

  2. Immunomodulatory Effects of Amblyomma variegatum Saliva on Bovine Cells: Characterization of Cellular Responses and Identification of Molecular Determinants

    Science.gov (United States)

    Rodrigues, Valérie; Fernandez, Bernard; Vercoutere, Arthur; Chamayou, Léo; Andersen, Alexandre; Vigy, Oana; Demettre, Edith; Seveno, Martial; Aprelon, Rosalie; Giraud-Girard, Ken; Stachurski, Frédéric; Loire, Etienne; Vachiéry, Nathalie; Holzmuller, Philippe

    2018-01-01

    The tropical bont tick, Amblyomma variegatum, is a tick species of veterinary importance and is considered as one of major pest of ruminants in Africa and in the Caribbean. It causes direct skin lesions, transmits heartwater, and reactivates bovine dermatophilosis. Tick saliva is reported to affect overall host responses through immunomodulatory and anti-inflammatory molecules, among other bioactive molecules. The general objective of this study was to better understand the role of saliva in interaction between the Amblyomma tick and the host using cellular biology approaches and proteomics, and to discuss its impact on disease transmission and/or activation. We first focused on the immuno-modulating effects of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages in vitro. We analyzed its immuno-suppressive properties by measuring the effect of saliva on PBMC proliferation, and observed a significant decrease in ConA-stimulated PBMC lymphoproliferation. We then studied the effect of saliva on bovine macrophages using flow cytometry to analyze the expression of MHC-II and co-stimulation molecules (CD40, CD80, and CD86) and by measuring the production of nitric oxide (NO) and pro- or anti-inflammatory cytokines. We observed a significant decrease in the expression of MHC-II, CD40, and CD80 molecules, associated with decreased levels of IL-12-p40 and TNF-α and increased level of IL-10, which could explain the saliva-induced modulation of NO. To elucidate these immunomodulatory effects, crude saliva proteins were analyzed using proteomics with an Orbitrap Elite mass spectrometer. Among the 336 proteins identified in A. variegatum saliva, we evidenced bioactive molecules exhibiting anti-inflammatory, immuno-modulatory, and anti-oxidant properties (e.g., serpins, phospholipases A2, heme lipoprotein). We also characterized an intriguing ubiquitination complex that could be involved in saliva

  3. Melatonin improves the quality of in vitro produced (IVP bovine embryos: implications for blastocyst development, cryotolerance, and modifications of relevant gene expression.

    Directory of Open Access Journals (Sweden)

    Feng Wang

    Full Text Available To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10(-7 M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10(-7 M significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.

  4. Successful transplantation of in vitro expanded human cadaver corneal endothelial precursor cells on to a cadaver bovine's eye using a nanocomposite gel sheet.

    Science.gov (United States)

    Parikumar, Periyasamy; Haraguchi, Kazutoshi; Ohbayashi, Akira; Senthilkumar, Rajappa; Abraham, Samuel J K

    2014-05-01

    In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine's eyes. HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes', aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies. Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible. The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.

  5. Receptor stimulated formation of inositol phosphates in cultures of bovine adrenal medullary cells: the effects of bradykinin, bombesin and neurotensin.

    Science.gov (United States)

    Bunn, S J; Marley, P D; Livett, B G

    1990-04-01

    The ability of a number of drugs and neuropeptides to stimulate phosphoinositide metabolism in cultured bovine adrenal medullary cells has been assessed. Low concentrations (10 nM) of angiotensin II, bradykinin, histamine, arginine-vasopressin, and bombesin, and high (10 microM) concentrations of oxytocin, prostaglandins E1, and E2, beta-endorphin, and neurotensin stimulated significant accumulation of [3H]inositol phosphates in adrenal medullary cells preloaded with [3H)]inositol. Bradykinin stimulated a significant response at concentration as low as 10pM, with an EC50 of approximately 0.5 nM. The response was markedly inhibited by the bradykinin B2 antagonist [Thi5,8,D-Phe7] bradykinin but not the B1 antagonist [Des-Arg9,Leu8] bradykinin. Higher concentrations of bombesin and neurotensin were required to elicit a response (10 nM and 10 microM respectively). The bombesin response was sensitive to inhibition by the bombesin antagonist [D-Arg1,D-Pro2,D-Trp7,9Leu11]-substance P. In contrast, the neurotensin response was not reduced by the NT1 antagonist [D-Trp11]-neurotensin. These results indicate there are a number of agents that can stimulate phosphatidylinositide hydrolysis in the adrenal medullary cells by acting on different classes of receptors. Such a range of diverse agonists that stimulate inositol phosphate formation will facilitate further analysis of the phosphatidylinositide breakdown in chromaffin cell function.

  6. Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

    Science.gov (United States)

    Pan, W-R; Chen, P-W; Chen, Y-L S; Hsu, H-C; Lin, C-C; Chen, W-J

    2013-01-01

    Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 μM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Male bovine GH transgenic mice have decreased adiposity with an adipose depot-specific increase in immune cell populations.

    Science.gov (United States)

    Benencia, Fabian; Harshman, Stephanie; Duran-Ortiz, Silvana; Lubbers, Ellen R; List, Edward O; Householder, Lara; Al-Naeeli, Mawadda; Liang, Xiaoyu; Welch, Lonnie; Kopchick, John J; Berryman, Darlene E

    2015-05-01

    White adipose tissue (WAT) is composed of mature adipocytes and a stromal vascular fraction (SVF), which contains a variety of cells, including immune cells that vary among the different WAT depots. Growth hormone (GH) impacts immune function and adiposity in an adipose depot-specific manner. However, its effects on WAT immune cell populations remain unstudied. Bovine GH transgenic (bGH) mice are commonly used to study the in vivo effects of GH. These giant mice have an excess of GH action, impaired glucose metabolism, decreased adiposity, increased lean mass, and a shortened lifespan. Therefore, the purpose of this study was to characterize the WAT depot-specific differences in immune cell populations in the presence of excess GH in vivo. Three WAT depots were assessed: inguinal (sc), epididymal (EPI), and mesenteric (MES). Subcutaneous and MES bGH WAT depots showed a significantly higher number of total SVF cells, yet only MES bGH WAT had higher leukocyte counts compared with control samples. By means of flow cytometry analysis of the SVF, we detected greater macrophage and regulatory T-cell infiltration in sc and MES bGH WAT depots compared with controls. However, no differences were observed in the EPI WAT depot. RNA-sequencing confirmed significant alterations in pathways related to T-cell infiltration and activation in the sc depot with fewer significant changes in the EPI bGH WAT depot. These findings collectively point to a previously unrecognized role for GH in influencing the distribution of WAT immune cell populations in a depot-specific manner.

  8. IL-6 Promotes FSH-Induced VEGF Expression Through JAK/STAT3 Signaling Pathway in Bovine Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Meng Yang

    2017-11-01

    Full Text Available Background/Aims: Vascular endothelial growth factor (VEGF has been demonstrated to play a pivotal role in the regulation of angiogenesis in ovarian follicular development, particularly during the preovulatory period. Although numerous studies have shown that interleukin-6 (IL-6 is one of the major inducing factors that regulate the expression of VEGF in non-ovarian cells, whether it involved in regulating the expression of VEGF in normal ovarian granulosa cells is still unknown. The aim of this study was to elucidate the mechanisms underlying the effect of IL-6 on FSH-induced VEGF expression in bovine granulosa cells derived from large follicles. Methods: VEGF mRNA expression in granulosa cells after IL-6 with/without inhibitors treatment was analyzed by RT-qPCR. Phosphorylation levels of ERK1/2 and STAT3 proteins induced by IL-6 were analyzed by western blotting. The protein levels produced by granulosa cells were detected by ELISA. Results: High concentration of IL-6 (10ng/ml can significantly up-regulate FSH-induced VEGF gene and protein expression levels in granulosa cells, and also promote the VEGF upstream regulators HIF-1α and COX2 mRNA expression. VEGF expression levels were significantly decreased after specifically blocking HIF-1α and COX2 by using inhibitors. The up-regulation effect of IL-6 on FSH-induced VEGF expression in granulosa cells mainly through activating the JAK/STAT3 signaling pathway, which can be impaired by JAK inhibitors. Conclusion: IL-6 can promote FSH-induced VEGF expression in granulosa cells, which is mainly achieved by increasing the expression of HIF-1α and COX2.This promoting effect is mediated by activating the JAK/STAT3 pathway. Moreover, there may be a synergistic relationship between FSH and IL-6 in the regulation of VEGF expression.

  9. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

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    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  10. Antibacterial Activity of 7-Epiclusianone and Its Novel Copper Metal Complex on Streptococcus spp. Isolated from Bovine Mastitis and Their Cytotoxicity in MAC-T Cells.

    Science.gov (United States)

    de Barros, Mariana; Perciano, Pedro Griffo; Dos Santos, Marcelo Henrique; De Oliveira, Leandro Licursi; Costa, Éderson D'Martin; Moreira, Maria Aparecida Scatamburlo

    2017-05-17

    Mastitis is an inflammation of mammary gland parenchyma that adversely affects bovine health and dairy production worldwide despite significant efforts to eradicate it. The aim of this work was to characterize the antimicrobial activity of 7-epiclusianone (7-epi), a compound extracted from the Rheedia brasiliensis fruit, its complex with copper against Streptococcus spp. isolated from bovine mastitis, and to assess their cytotoxicity to bovine mammary alveolar cells (MAC-T). The complex 7-epiclusianone-Cu (7-epi-Cu) was an amorphous green solid with optical activity. Its vibrational spectrum in the infrared region showed absorption bands in the high-frequency region, as well as bands that can be attributed to the unconjugated and conjugated stretching of the free ligand. The complex was anhydrous. One of the tested bacterial strains was not sensitive to the compounds, while the other three had MIC values of 7.8 µg mL -1 and minimum bactericidal concentration (MBC) values between 15.6 and 31.3 µg mL -1 . These two compounds are bacteriostatic, did not cause damage to the cell wall and, at sub-inhibitory concentrations, did not induce bacterial adhesion. The compounds were not cytotoxic. Based on these results, 7-epi and 7-epi-Cu exhibited desirable antimicrobial properties and could potentially be used in bovine mastitis treatment.

  11. Self-Assembled Fluorescent Bovine Serum Albumin Nanoprobes for Ratiometric pH Measurement inside Living Cells.

    Science.gov (United States)

    Yang, Qiaoyu; Ye, Zhongju; Zhong, Meile; Chen, Bo; Chen, Jian; Zeng, Rongjin; Wei, Lin; Li, Hung-wing; Xiao, Lehui

    2016-04-20

    In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6-8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.

  12. Supramolecular assembly of biological molecules purified from bovine nerve cells: from microtubule bundles and necklaces to neurofilament networks

    International Nuclear Information System (INIS)

    Needleman, Daniel J; Jones, Jayna B; Raviv, Uri; Ojeda-Lopez, Miguel A; Miller, H P; Li, Y; Wilson, L; Safinya, C R

    2005-01-01

    With the completion of the human genome project, the biosciences community is beginning the daunting task of understanding the structures and functions of a large number of interacting biological macromolecules. Examples include the interacting molecules involved in the process of DNA condensation during the cell cycle, and in the formation of bundles and networks of filamentous actin proteins in cell attachment, motility and cytokinesis. In this proceedings paper we present examples of supramolecular assembly based on proteins derived from the vertebrate nerve cell cytoskeleton. The axonal cytoskeleton in vertebrate neurons provides a rich example of bundles and networks of neurofilaments, microtubules (MTs) and filamentous actin, where the nature of the interactions, structures, and structure-function correlations remains poorly understood. We describe synchrotron x-ray diffraction, electron microscopy, and optical imaging data, in reconstituted protein systems purified from bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories of polyelectrolyte bundling, including three-dimensional MT bundles and two-dimensional MT necklaces

  13. Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

    Science.gov (United States)

    Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd; Juhl, Morten; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

    2017-01-01

    Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  14. RNAi-mediated knockdown of MTNR1B without disrupting the effects of melatonin on apoptosis and cell cycle in bovine granulose cells

    Directory of Open Access Journals (Sweden)

    Wenju Liu

    2018-04-01

    Full Text Available Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3, decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL and anti-oxidant genes (SOD1 and GPX4 without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.

  15. Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection.

    Science.gov (United States)

    Suzuki, Saori; Konnai, Satoru; Okagawa, Tomohiro; Ikebuchi, Ryoyo; Nishimori, Asami; Kohara, Junko; Mingala, Claro N; Murata, Shiro; Ohashi, Kazuhiko

    2015-02-15

    Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4(+) T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection. Copyright © 2014 Elsevier B.V. All rights

  16. Cumulus Kuopion työmatkustajien asiakastyytyväisyys

    OpenAIRE

    Lampinen, Miia; Lamminmäki, Roosa-Maria

    2015-01-01

    Eri hotelliryhmissä asiakkaat erotellaan heidän tarpeidensa ja toiveidensa mukaan. Asiakas on se, joka määrittelee yrityksen laadun. Business-asiakkaat valitsevat usein kansallisen tai kansainvälisen ketjuhotellin, joka täyttää tietyt laatustandardit. Yleensä he myös sitoutuvat ketjun kanta-asiakasohjelmiin. Kanta-asiakasjärjestelmät sisältävät yleensä edullisempien majoitusten lisäksi muitakin etuja. Cumulus Kuopio on hotelli hyvien kulkuyhteyksien päässä, aivan Kuopion rautatieaseman vieres...

  17. α-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. II. Identification using a sequence-specific monoclonal antibody

    International Nuclear Information System (INIS)

    Kobrin, M.S.; Samsoondar, J.; Kudlow, J.E.

    1986-01-01

    Untransformed bovine anterior pituitary cells cultured in serum-free defined medium secrete an epidermal growth factor (EGF)-like peptide with an amino acid composition similar to rat or human α-transforming growth factor (αTGF). To further characterize the bovine pituitary αTGF, it was compared to a human αTGF partially purified from the conditioned medium of a human melanoma cell line. An anti-αTGF monoclonal antibody, MF9, was produced from hybridomas derived from mice immunized with a 17-residue synthetic peptide corresponding to the carboxyl-terminal sequence of rat αTGF. The hybridoma supernatants were initially screened for the ability to immunoprecipitate 125 I-peptide and then tested for recognition of human αTGF. Only 2 of 36 antipeptide antibodies recognized the native αTGF. The binding of 125 I-peptide to MF9 was displaced by human αTGF but not by EGF. Bovine pituitary αTGF also displaced the binding of 125 I-peptide to MF9 in a similar manner to human αTGF. Both iodinated human and bovine pituitary αTGF were immunoprecipitated by MF9 whereas 125 I-EGF was not. Tryptic digests of both 125 I-αTGFs chromatographed to give a single, indistinguishable peak of iodinated material on a reverse-phase C 18 high performance liquid chromatography column when eluted with two different solvent systems, suggesting the generation of a single and identical tyrosine-containing tryptic peptide from both αTGFs. The comparisons of the bovine pituitary and human melanoma αTGF using a sequence-specific monoclonal antibody and peptide mapping suggest that these αTGFs are related and that αTGF production is not limited to transformed or fetal sources

  18. Oleic acid induces specific alterations in the morphology, gene expression and steroid hormone production of cultured bovine granulosa cells.

    Science.gov (United States)

    Yenuganti, Vengala Rao; Viergutz, Torsten; Vanselow, Jens

    2016-06-01

    After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Enhanced bovine serum albumin absorption on the N-hydroxysuccinimide activated graphene oxide and its corresponding cell affinity.

    Science.gov (United States)

    Xiong, Kun; Fan, Qingbo; Wu, Tingting; Shi, Haishan; Chen, Lin; Yan, Minhao

    2017-12-01

    By successively reacting with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the carboxyl on the graphene oxide (GO) surface was successfully activated into NHS active ester. In this study, bovine serum albumin (BSA) was selected as a model protein, used for studying the protein absorption capacity of the NHS activated GO (GO-EDC-NHS). Approximately 12.75mg of BSA could be covalent bonded onto the GO-EDC-NHS surface (BSA-CB-GO), whereas only 6.83mg of BSA physical absorbed onto the GO surface (BSA-NB-GO). With a 168h of phosphate buffer saline (PBS) soaking, the BSA accumulative desorption ratio, which was accordingly assigned to the BSA-NB-GO and the BSA-CB-GO, was separately 29.91wt% and 2.95wt%. Consequently, it proved GO-EDC-NHS exhibited more stable and stronger BSA absorption capacity. As compared to the mouse bone marrow mesenchymal stem cells (mBMSCs) cultivated on the BSA-NB-GO surface, the immunofluorescence staining images showed that more vinculins and integrin α5 were visible in the mBMSCs cultivated on the BSA-CB-GO surface, they also produced more distinct stress fibers and actin-containing microfilaments. In summary, BSA-CB-GO possesses an excellent cell affinity, which can be considered as a promising functional material used for promoting the bone remodeling. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Effects of selected endocrine disruptors on meiotic maturation, cumulus expansion, syntesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes

    Czech Academy of Sciences Publication Activity Database

    Mlynarčíková, A.; Nagyová, Eva; Ficková, M.; Scsuková, S.

    2009-01-01

    Roč. 23, - (2009), s. 371-377 ISSN 0887-2333 R&D Projects: GA ČR GA305/05/0960 Grant - others:VEGA(SK) 2/6171/26; EU(XE) QLK4-CT-2002-02637 Institutional research plan: CEZ:AV0Z50450515 Keywords : oocyte-cumulus complex * meiotic maturation * cumulus expansion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.060, year: 2009

  1. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

    DEFF Research Database (Denmark)

    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre...... embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI...

  2. Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG

    Directory of Open Access Journals (Sweden)

    Jacques G. Lussier

    2017-11-01

    Full Text Available Abstract Background Ovulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC of ovulatory follicles. Methods Granulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF and from ovulatory follicles (OF obtained 24 h following injection of human chorionic gonadotropin (hCG. A granulosa cell subtracted cDNA library (OF-DF was generated using suppression subtractive hybridization and screened. Results Detection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression. Conclusions We conclude that we have identified

  3. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    International Nuclear Information System (INIS)

    Yashima, N.; Wada, A.; Izumi, F.

    1986-01-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of 45 Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of 45 Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of 45 Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the 45 Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the 45 Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of 45 Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of 45 Ca. Based on these findings, the authors suggest that inhibition of the 45 Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels

  4. In vivo effects of human adipose-derived stem cells reseeding on acellular bovine pericardium in nude mice.

    Science.gov (United States)

    Wu, Qingkai; Dai, Miao; Xu, Peirong; Hou, Min; Teng, Yincheng; Feng, Jie

    2016-01-01

    Tissue-engineered biologic products may be a viable option in the reconstruction of pelvic organ prolapse (POP). This study was based on the hypothesis that human adipose-derived stem cells (hASCs) are viable in acellular bovine pericardium (ABP), when reseeded by two different techniques, and thus, aid in the reconstruction. To investigate the reseeding of hASCs on ABP grafts by using non-invasive bioluminescence imaging (BLI), and to identify the effective hASCs-scaffold combinations that enabled regeneration. Thirty female athymic nude mice were randomly divided into three groups: In the VIVO group, ABPs were implanted in the subcutaneous pockets and enhanced green fluorescent protein luciferase (eGFP·Luc)-hASCs (1 × 10(6) cells/50 µL) were injected on the ABP at the same time. In the VITRO group, the mice were implanted with grafts that ABP were co-cultured with eGFP·Luc-hASCs in vitro. The BLANK group mice were implanted with ABP only. The eGFP·Luc-hASCs reseeded on ABP were analyzed by BLI, histology, and immunohistochemistry. The eGFP·Luc-hASCs reseeded on ABP could be visualized at 12 weeks in vivo. Histology revealed that the VIVO group displayed the highest cell ingrowths, small vessels, and percent of collagen content per unit area. Desmin and α-smooth muscle actin were positive at the same site in the VIVO group cells. However, few smooth muscles were observed in the VITRO and BLANK groups. These results suggest that hASCs reseeded on ABP in vivo during surgery may further enhance the properties of ABP and may promote regeneration at the recipient site, resulting in a promising treatment option for POP. © 2016 by the Society for Experimental Biology and Medicine.

  5. MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway.

    Science.gov (United States)

    Pande, Hari Om; Tesfaye, Dawit; Hoelker, Michael; Gebremedhn, Samuel; Held, Eva; Neuhoff, Christiane; Tholen, Ernst; Schellander, Karl; Wondim, Dessie Salilew

    2018-05-01

    The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches. The role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling

  6. Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture.

    Science.gov (United States)

    Shimizu, Takashi; Kaji, Ayami; Murayama, Chiaki; Magata, Fumie; Shirasuna, Koumei; Wakamiya, Kaori; Okuda, Kiyoshi; Miyamoto, Akio

    2012-01-01

    Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48 h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48 h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Effects of elevated glucose concentration on cultured bovine retinal endothelial (BRE) cells

    International Nuclear Information System (INIS)

    Capetandes, A.; Gerritsen, M.E.

    1986-01-01

    Salient clinical features of diabetic retinopathy include capillary microaneurysm and neovascularization, which progress with the severity of the disease. It has been suggested that exposure of the retinal vascular cells to high glucose concentrations may play a causative role in the retinopathy. In the present study, the effects of variant media glucose concentrations on BRE cell growth were determined. Normal growth curves were obtained with glucose concentrations of 100, 450 and 600 mg%, but the replication rate was decreased with 600 mg%. To determine if elevated glucose concentrations also altered DNA synthesis, BRE cells cultivated with 100 and 600 mg% glucose demonstrated increased thymidine uptake and total DNA content compared to the 100 mg% group. Furthermore, vacuolation and increased cell diameter occurred in BRE cells cultivated 600 mg% compared to 100 mg% glucose. In conclusion, increases in media glucose concentrations result in a decreased cellular replication rate, increased DNA synthesis and increased cell diameter during the log phase of growth

  8. Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

    International Nuclear Information System (INIS)

    Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W.

    2007-01-01

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB

  9. The effect of oxygen on aldosterone release from bovine adrenocortical cells in vitro: PO2 versus steroidogenesis.

    Science.gov (United States)

    Raff, H; Kohandarvish, S

    1990-08-01

    Hypoxia decreases plasma aldosterone in vivo without a decrease in PRA, angiotensin II (ANG II), ACTH, or cortisol. The present study evaluated whether this could be due to a direct, specific inhibitory effect on the zona glomerulosa related to the magnitude of the decrease in oxygen (O2). Bovine adrenocortical cells were dispersed with collagenase and studied in vitro within 48 h. Cells were stimulated for 2 h with ANG II (0.1-1000 nM) or (Bu)2cAMP (0.3-3 mM) under oxygen levels ranging from 0 to 100% O2 (PO2 from 66 +/- 4 to 561 +/- 46 torr) vs. a reference gas mixture (21% O2 PO2 approximately 140 torr). Exposure to 123 +/- 8, 110 +/- 12, 100 +/- 16, and 66 +/- 4 torr led to 27%, 30%, 40% and 70% inhibition, respectively, of 3 nM ANG II-stimulated aldosterone secretion as compared to 140 +/- 16 torr (reference). Exposure to hyperoxia (288 +/- 36 to 561 +/- 46 torr) led to a small (10%) increase in ANG II-stimulated aldosterone secretion which was not statistically significant. The P50 (half-maximal PO2) for aldosteronogenesis was approximately 95 torr. The results for other doses of ANG II and for cAMP were similar. The inhibitory effect of low O2 was reversed by returning the cells to reference conditions (140 +/- 16 torr). Cortisol secretion was not significantly affected by changes in oxygen tension. We conclude that small changes in O2 within the physiological range directly and specifically inhibit aldosteronogenesis in a dose-dependent manner with a P50 of approximately 95 torr. Inhibition of cAMP-stimulated aldosterone secretion suggests a postreceptor site of action. This direct, reversible, and specific effect on the zona glomerulosa of the adrenal cortex may account for the dissociation of renin and aldosterone during hypoxia in vivo.

  10. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Fani, Nesa [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ziadlou, Reihane [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahhoseini, Maryam, E-mail: m.shahhoseini@royaninstitute.org [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Baghaban Eslaminejad, Mohamadreza, E-mail: eslami@royaninstitute.org [Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of)

    2016-06-10

    Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

  11. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

    International Nuclear Information System (INIS)

    Fani, Nesa; Ziadlou, Reihane; Shahhoseini, Maryam; Baghaban Eslaminejad, Mohamadreza

    2016-01-01

    Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

  12. Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

    Directory of Open Access Journals (Sweden)

    Yu-Ting Huang

    2007-12-01

    Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

  13. Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

    Science.gov (United States)

    Boarder, M R; Plevin, R; Marriott, D B

    1988-10-25

    The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

  14. Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

    International Nuclear Information System (INIS)

    De Lean, A.; Cantin, M.

    1986-01-01

    The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levels of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with [ 32 P] NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production

  15. Differences in fetal bovine serum affect the responsiveness of cells to mechanical loads

    NARCIS (Netherlands)

    Rubert, M.; Vetsch, J.R.; Storz, L.; Lehtoviita, I.; Mueller, R.M.; Hofmann, S.

    2015-01-01

    Nowadays, the end-point of a cell culture in bone tissue engineering (BTE) is the acquisition of a well mineralized extracellular matrix. The biological performance of BTE relies on evaluation of the cell capacity to proliferate and to produce extracellular matrix by quantification of gene

  16. Polyfunctional cytokine responses by central memory CD4+T cells in response to bovine tuberculosis

    Science.gov (United States)

    CD4 T cells are crucial in immunity to tuberculosis (TB). Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) and play relevant roles in several chronic infections, including human TB and HIV. Mycobacterium ...

  17. Polyfunctional cytokine responses by central memory CD4*T cells in response to bovine tuberculosis

    Science.gov (United States)

    CD4 T cells are crucial in immunity to tuberculosis (TB). Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) and play relevant roles in several chronic infections, including human TB. Mycobacterium bovis in...

  18. Effect of cysteine supplementation on in vitro maturation of bovine ...

    African Journals Online (AJOL)

    Parham

    2011-11-09

    Nov 9, 2011 ... or more layer of cumulus cells and homogeneous granular ooplasm were selected for IVM procedures (Badr, 2009). In vitro maturation of oocytes. The basic medium for IVM was HEPES-buffered tissue culture medium 199 supplemented with 0.2 mM sodium pyruvate, 1 µg/ml. 17-β-estradiol, 10% fetal calf ...

  19. Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets

    Energy Technology Data Exchange (ETDEWEB)

    Fell, A.H.; Preston, P.M. (Edinburgh Univ. (United Kingdom))

    1992-07-01

    Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. (Author).

  20. Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets

    International Nuclear Information System (INIS)

    Fell, A.H.; Preston, P.M.

    1992-01-01

    Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. (Author)

  1. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    Directory of Open Access Journals (Sweden)

    Betty R Liu

    Full Text Available Cell-penetrating peptides (CPPs have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW from bovine lactoferricin (LFcin, stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  2. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    Science.gov (United States)

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  3. Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

    Directory of Open Access Journals (Sweden)

    Piccinato Carla A

    2012-11-01

    Full Text Available Abstract Background Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE in the steroid hormone profile of a serum-free granulosa cell (GC culture system in the context of follicular development and dominance. Methods Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7 M resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8 M, a non-selective beta-adrenergic antagonist. Conclusions The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

  4. Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions.

    Science.gov (United States)

    Piccinato, Carla A; Montrezor, Luis H; Collares, Cristhianna A V; Vireque, Alessandra A; Rosa e Silva, Alzira A M

    2012-11-22

    Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose-response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone production was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

  5. Transcriptome dynamics of human pluripotent stem cell-derived contracting cardiomyocytes using an embryoid body model with fetal bovine serum.

    Science.gov (United States)

    Jung, Kwang Bo; Son, Ye Seul; Lee, Hana; Jung, Cho-Rok; Kim, Janghwan; Son, Mi-Young

    2017-07-25

    Cardiomyocyte (CM) differentiation techniques for generating adult-like mature CMs remain imperfect, and the plausible underlying mechanisms remain unclear; however, there are a number of current protocols available. Here, to explore the mechanisms controlling cardiac differentiation, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human pluripotent stem cells (hPSCs) into CMs using embryoid body (EB) formation. We optimized and updated the protocol to efficiently generate contracting CMs from hPSCs by adding fetal bovine serum (FBS) as a medium supplement, which could have a significant impact on the efficiency of cardiac differentiation. To identify genes, biological processes, and pathways involved in the cardiac differentiation of hPSCs, integrative and comparative analyses of the transcriptome profiles of differentiated CMs from hPSCs and of control CMs of the adult human heart (CM-AHH) were performed using gene ontology, functional annotation clustering, and pathway analyses. Several genes commonly regulated in the differentiated CMs and CM-AHH were enriched in pathways related to cell cycle and nucleotide metabolism. Strikingly, we found that current differentiation protocols did not promote sufficient expression of genes involved in oxidative phosphorylation to differentiate CMs from hPSCs compared to the expression levels in CM-AHH. Therefore, to obtain mature CMs similar to CM-AHH, these deficient pathways in CM differentiation, such as energy-related pathways, must be augmented prior to use for in vitro and in vivo applications. This approach opens up new avenues for facilitating the utilization of hPSC-derived CMs in biomedical research, drug evaluation, and clinical applications for patients with cardiac failure.

  6. A Heuristic Parameterization for the Integrated Vertical Overlap of Cumulus and Stratus

    Science.gov (United States)

    Park, Sungsu

    2017-10-01

    The author developed a heuristic parameterization to handle the contrasting vertical overlap structures of cumulus and stratus in an integrated way. The parameterization assumes that cumulus is maximum-randomly overlapped with adjacent cumulus; stratus is maximum-randomly overlapped with adjacent stratus; and radiation and precipitation areas at each model interface are grouped into four categories, that is, convective, stratiform, mixed, and clear areas. For simplicity, thermodynamic scalars within individual portions of cloud, radiation, and precipitation areas are assumed to be internally homogeneous. The parameterization was implemented into the Seoul National University Atmosphere Model version 0 (SAM0) in an offline mode and tested over the globe. The offline control simulation reasonably reproduces the online surface precipitation flux and longwave cloud radiative forcing (LWCF). Although the cumulus fraction is much smaller than the stratus fraction, cumulus dominantly contributes to precipitation production in the tropics. For radiation, however, stratus is dominant. Compared with the maximum overlap, the random overlap of stratus produces stronger LWCF and, surprisingly, more precipitation flux due to less evaporation of convective precipitation. Compared with the maximum overlap, the random overlap of cumulus simulates stronger LWCF and weaker precipitation flux. Compared with the control simulation with separate cumulus and stratus, the simulation with a single-merged cloud substantially enhances the LWCF in the tropical deep convection and midlatitude storm track regions. The process-splitting treatment of convective and stratiform precipitation with an independent precipitation approximation (IPA) simulates weaker surface precipitation flux than the control simulation in the tropical region.

  7. Covalent transfer of heavy chains of inter-alpha-trypsin inhibitor family proteins to hyaluronan in in vivo and in vitro expanded porcine oocyte-cumulus complexes

    Czech Academy of Sciences Publication Activity Database

    Nagyová, Eva; Camaioni, A.; Procházka, Radek; Salustri, A.

    2004-01-01

    Roč. 71, - (2004), s. 1838-1843 ISSN 0006-3363 R&D Projects: GA AV ČR IAA5045102; GA ČR GA523/04/0574; GA AV ČR KSK5052113 Institutional research plan: CEZ:AV0Z5045916 Keywords : cumulus cells * fertilization * follicle-stimulating Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.550, year: 2004

  8. Proliferation Rates of Bovine Primary Muscle Cells Relate to Liveweight and Carcase Weight in Cattle

    Science.gov (United States)

    Coles, Chantal A.; Wadeson, Jenny; Leyton, Carolina P.; Siddell, Jason P.; Greenwood, Paul L.; White, Jason D.; McDonagh, Matthew B.

    2015-01-01

    Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5–20 hours in culture) in vitro (P cattle (P cattle (P cattle. PMID:25875203

  9. In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1.

    Science.gov (United States)

    Oliveira, C S; de Souza, M M; Saraiva, N Z; Tetzner, T A D; Lima, M R; Lopes, F L; Garcia, J M

    2012-06-01

    Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. © 2011 Blackwell Verlag GmbH.

  10. Differential responses of cells from human skin keratinocyte and bovine mammary epithelium to attack by pore-forming Staphylococcus aureus alpha-toxin.

    Science.gov (United States)

    Suriyaphol, Gunnaporn; Sarikaputi, Meena; Suriyaphol, Prapat

    2009-11-01

    Human skin keratinocytes HaCat attacked by Staphylococcus aureus alpha-toxin showed a transient drop of cellular ATP levels whereas in toxin-perforated bovine mammary epithelial cells (BMEC), the ATP levels dropped more slowly. Morphologically, during the ATP level depletion, HaCat cell developed a spacious intracellular vacuole together with the transient influx of trypan blue. WST-1 signal, which tested the function of mitochondrial enzyme in viable cells, also decreased concomitantly. On the other hand, BMEC excluded trypan blue and vacuolation was not observed throughout the experiment. We conclude that mammary epithelial cells resist the toxin better than keratinocytes. This is the first report showing that alpha-toxin enhances transient membrane permeability to large molecules, temporary vacuole formation and the transient defect of mitochondrial enzyme in viable cells without cell lysis.

  11. Evaluation of royal jelly as an alternative to fetal bovine serum in cell ...

    African Journals Online (AJOL)

    Background: Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth ... In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was ...

  12. Simplification of Bovine Somatic Cell Nuclear Transfer by Application of a Zona-Free Manipulation Technique

    DEFF Research Database (Denmark)

    Booth, Paul J; Tan, Shijian; Reipurth, Rikke

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The techni......Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods....... The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development......-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20...

  13. Development and characterization of a bovine monocyte-derived macrophage cell line

    Science.gov (United States)

    Monocytes circulate in the blood, and later differentiate into macrophages in the tissues. They are components of the innate arm of the immune response and are one of the first lines of defense again invading pathogens. However, they also serve as host cells for intracellular pathogens such as Mycob...

  14. Effect of bovine pellucid zone 3 monoclonal antibodies on B cell lymphoma 2 expressions of granulosa cell and mice (Mus musculus follicle diameter

    Directory of Open Access Journals (Sweden)

    Heti Ira Ayue

    2017-01-01

    Full Text Available Objective: To evaluate the effects of pellucid zone 3 monoclonal antibodies against B-cell lymphoma 2 (BCL-2 expression and mice follicle diameter at various time periods.Methods: The animal model of this study was 36 Balb/c mice (Mus musculus. A true experimental design was used with a post-test only control group approach. BCL-2 expression was observed using immunohistochemistry, while the follicle diameter was observed by haematoxylin-eosin staining. The data was analyzed using nested ANOVA to compare the results of the mean expression of BCL-2 on the 5th and 20th day of observation in the pre-antral and antral follicle between the control and treatment groups.Results: No significant differences were found in BCL-2 gene expression. There were also no significant differences in BCL-2 expression on the 10th day of pre-antral follicle analysis. Moreover, there were no significant differences between the mean follicle diameter on the 5th, 10th, and 20th day of pre-antral and antral follicle development between the control and treatment groups. The addition of bovine pellucid zone 3 (bZP3 monoclonal antibodies on the 5th and 20th day of observation did not decrease the expression of BCL-2 gene in the pre-antral and antral follicle of mice. Administering bZP3 monoclonal antibodies on the 10th day of observation did not affect BCL-2 expression in the pre-antral follicle but did decrease BCL-2 expression in the antral follicle. Supplying bZP3 monoclonal antibodies on the 5th, 10th and 20th day did not affect the diameter of pre-antral and antral follicles of the mice.Conclusion: The monoclonal antibodies bovine zona pelusida 3 has the potential to be developed as a safe immunocontraception preparation.

  15. Bovine leukemia virus reduces anti-viral cytokine activities and NK cytotoxicity by inducing TGF-β secretion from regulatory T cells.

    Science.gov (United States)

    Ohira, Kosuke; Nakahara, Ayako; Konnai, Satoru; Okagawa, Tomohiro; Nishimori, Asami; Maekawa, Naoya; Ikebuchi, Ryoyo; Kohara, Junko; Murata, Shiro; Ohashi, Kazuhiko

    2016-03-01

    CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-β(+) cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-β from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.

  16. All-trans retinoic acid increases the expression of oxidative myosin heavy chain through the PPARδ pathway in bovine muscle cells derived from satellite cells.

    Science.gov (United States)

    Kim, Jongkyoo; Wellmann, Kimberly B; Smith, Zachary K; Johnson, Bradley J

    2018-04-24

    All-trans retinoic acid (ATRA) has been associated with various physiological phenomenon in mammalian adipose tissue and skeletal muscle. We hypothesized that ATRA may affect skeletal muscle fiber type in bovine satellite cell culture through various transcriptional processes. Bovine primary satellite cell (BSC) culture experiments were conducted to determine dose effects of ATRA on expression of genes and protein levels related to skeletal muscle fiber type and metabolism. The semimembranosus from crossbred steers (n = 2 steers), aged approximately 24 months, were used to isolate BSC for 3 separate assays. Myogenic differentiation was induced using 3% horse serum upon cultured BSC with increasing doses (0, 1, 10, 100, 1000 nM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of protein kinase B (Akt), AMP-activated protein kinase alpha (AMPK), glucose transporter 4 (GLUT4), myogenin, lipoprotein lipase (LPL), myosin heavy chain (MHC) I, MHC IIA,MHC IIX, insulin like growth factor -1 (IGF-1), Peroxisome proliferator activated receptor gamma (PPARγ), PPARδ, and Smad transcription factor 3 (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). The mRNA expression of LPL was increased (P < 0.05) with 100 and 1000nM of ATRA. Expression of GLUT4 was altered (P < 0.05) by ATRA. The treatment of ATRA (1000nM) also increased (P < 0.05) mRNA gene expression of SMAD3. The gene expression of both PPARδ and PPARγ were increased (P < 0.05) with 1000nM of ATRA. Protein level of PPARδ was also affected (P < 0.05) by 1000nM of ATRA and resulted in a greater (P < 0.05) protein level of PPARδ compared to CON. All-trans retinoic acid (10nM) increased gene expression of MHC I (P < 0.05) compared to CON. Expression of MHC IIA was also influenced (P < 0.05) by ATRA. The mRNA expression of MHC IIX was decreased (P < 0.05) with 100 and 1000nM of ATRA.In muscle cells, ATRA may cause muscle fibers to transition towards the MHC

  17. Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

    Science.gov (United States)

    Accogli, Gianluca; Douet, Cécile; Ambruosi, Barbara; Martino, Nicola Antonio; Uranio, Manuel Filioli; Deleuze, Stefan; Dell'Aquila, Maria Elena; Desantis, Salvatore; Goudet, Ghylène

    2014-12-01

    Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. © 2014 Wiley Periodicals, Inc.

  18. Evaluating The Effect of Melatonin on HAS2, and PGR expression, as well as Cumulus Expansion, and Fertility Potential in Mice.

    Science.gov (United States)

    Ezzati, Maryam; Roshangar, Leila; Soleimani Rad, Jafar; Karimian, Nahid

    2018-04-01

    Infertility is a worldwide health problem which affects approximately 15% of sexually active couples. One of the factors influencing the fertility is melatonin. Also, protection of oocytes and embryos from oxidative stress inducing chemicals in the culture medium is important. The aim of the present study was to investigate if melatonin could regulate hyaluronan synthase-2 (HAS2) and Progesterone receptor (PGR) expressions in the cumulus cells of mice oocytes and provide an in vitro fertilization (IVF) approach. In this experimental study, for this purpose, 30 adult female mice and 15 adult male mice were used. The female mice were superovulated using 10 U of pregnant mare serum gonadotropin (PMSG) and 24 hours later, 10 U of human chorionic gonadotropin (hCG) were injected. Next, cumulus oocyte complexes (COCs) were collected from the oviducts of the female mice by using a matrix-flushing method. The cumulus cells were cultured with melatonin 10 μM for 6 hours and for real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for evaluation of HAS2 and PGR expression levels. The fertilization rate was evaluated through IVF. All the data were analyzed using a t test. The results of this study showed that HAS2 and PGR expressions in the cumulus cells of the mice receiving melatonin increased in comparison to the control groups. Also, IVF results revealed an enhancement in fertilization rate in the experimental groups compared to the control groups. To improve the oocyte quality and provide new approaches for infertility treatment, administration of melatonin as an antioxidant, showed promising results. Thus, it is concluded that fertility outcomes can be improved by melatonin it enhances PGR. Copyright© by Royan Institute. All rights reserved.

  19. Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Cheek, T R; Thastrup, Ole

    1989-01-01

    and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from......-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)...

  20. Biochemical and topological analysis of bovine sperm cells induced by low power laser irradiation

    Science.gov (United States)

    Dreyer, T. R.; Siqueira, A. F. P.; Magrini, T. D.; Fiorito, P. A.; Assumpção, M. E. O. A.; Nichi, M.; Martinho, H. S.; Milazzotto, M. P.

    2011-07-01

    Low-level laser irradiation (LLLI) increases ATP production and energy supply to the cell which could increase sperm motility, acrossomal reaction and consequently the fertilizing potential. The aim of this study was to characterize the biochemical and topological changes induced by low power laser irradiation on bull sperm cells. Post-thawing sperm were irradiated with a 633nm laser with fluence rates of 30, 150 and 300mJ.cm-2 (power of 5mW for 1, 5 and 10minutes, respectively); 45, 230, and 450mJ.cm-2 (7.5mW for 1, 5 and 10 minutes); and 60, 300 and 600mJ.cm-2 (10mW for 1, 5 and 10 minutes). Biochemical and metabolical changes were analyzed by FTIR and flow cytometry; oxygen reactive species production was assessed by TBARS and the morphological changes were evaluated by AFM. Motility had no difference among times or powers of irradiation. Increasing in ROS generation was observed with power of 5mW compared to 7.5 and 10mW, and with 10min of irradiation in comparison with 5 and 1min of irradiation. This higher ROS generation was related to an increase in acrossomal and plasma membrane damage. FTIR results showed that the amount of lipids was inversely proportional to the quantity of ROS generated. AFM images showed morphological differences in plasma/acrossomal membrane, mainly on the equatorial region. We conclude that LLLI is an effective method to induce changes on sperm cell metabolism but more studies are necessary to establish an optimal dose to increase the fertility potential of these cells.

  1. Influence of bradykinin on diacylglycerol and phosphatidic acid accumulation in cultured bovine adrenal chromaffin cells.

    Science.gov (United States)

    Owen, P J; Boarder, M R

    1991-09-01

    Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate

    DEFF Research Database (Denmark)

    Jensen, Simon S; Gruber, Reinhard; Buser, Daniel

    2015-01-01

    OBJECTIVES: The occurrence of multinucleated giant cells (MNGCs) on bone substitute materials has been recognized for a long time. However, there have been no studies linking material characteristics with morphology of the MNGCs. The aim was to analyze the qualitative differences of MNGCs on two ...... osteoclasts. CONCLUSION: MNGCs demonstrated distinctly different histological features depending on the bone substitute material used. Further research is warranted to understand the clinical implications of these morphological observations....

  3. Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function

    NARCIS (Netherlands)

    Koets, A.; Rutten, V.; Hoek, van A.; Mil, van F.; Muller, K.; Bakker, D.; Gruys, E.; Eden, van W.

    2002-01-01

    Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically

  4. Cellular dynamics of bovine aortic smooth muscle cells measured using MEMS force sensors

    Science.gov (United States)

    Tsukagoshi, Takuya; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Takahashi, Hidetoshi; Matsumoto, Kiyoshi; Shimoyama, Isao

    2018-04-01

    Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (~2.5 µs) over long periods (~3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.

  5. Fuel feeds function: Energy balance and bovine peripheral blood mononuclear cell activation.

    Science.gov (United States)

    Schwarm, A; Viergutz, T; Kuhla, B; Hammon, H M; Schweigel-Röntgen, M

    2013-01-01

    A general phenomenon in peripartum mammals is the breakdown of (acquired) immunity. The incidence of parasite load, disease and inflammation often rise during the specific energetically demanding time of pregnancy and lactation. In this period, blood leukocytes display decreased DNA synthesis in response to mitogens in vitro. Leukocyte activation, the phase of the cell cycle preceding the DNA synthetic phase has hardly been investigated, but the few studies suggest that leukocyte activation may also be impaired by the limited energy/nutrient availability. Leukocyte activation is characterized by manifold processes, thus, we used the cellular oxygen consumption rate (OCR) as a measure of ATP turnover to support all these processes. We hypothesized that the activation of peripheral blood mononuclear cells (PBMC) - in terms of oxygen consumed over basal levels after in vitro stimulation - is altered by energy balance around parturition. We studied peripartum high-yielding dairy cows because they undergo substantial fluctuations in energy intake, energy output and body fat mass. We established a fluorescence-based test strategy allowing for long-term (≥24h) quantification of O(2)-consumption and studied the peripartum period from 5 weeks ante partum to 5 weeks postpartum. In addition, we determined cellular lactate production, DNA/RNA synthesis and cell size and zoo-technical parameters such as animal energy intake and milk yield were assessed, as well as selected plasma parameters, e.g. glucose concentration. The basal OCR of PBMC from pregnant, non-lactating cows (n=6, -5 weeks ante partum) was 1.19±0.15 nmol min(-1) (10(7)cells)(-1) and increased to maximum levels of 2.54±0.49 nmol min(-1) (10(7)cells)(-1) in phytohemagglutinin (PHA)-stimulated PBMC. The basal OCR did not change over the peripartum period. Whereas the activation indices, herein defined as the PHA-induced 24h-increase of OCR above baseline, amounted to 1.1±0.3, 4.2±0.3, 4.1±1.1, 2.1±0.3, and

  6. Bovine lactoferricin induces caspase-independent apoptosis in human B-lymphoma cells and extends the survival of immune-deficient mice bearing B-lymphoma xenografts.

    Science.gov (United States)

    Furlong, Suzanne J; Mader, Jamie S; Hoskin, David W

    2010-06-01

    Although current treatments based on the use of B-cell-specific anti-CD20 monoclonal antibodies and aggressive combinatorial chemotherapy have improved the survival of patients suffering from B-cell non-Hodgkin's lymphoma (NHL), some individuals fail to respond to treatment and relapses remain common. New and more effective treatments for B-cell NHL are therefore required. Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that is cytotoxic for several human tumor cell lines but does not harm healthy cells. Here we show that in vitro treatment with LfcinB caused Raji and Ramos human B-lymphoma cells to die by apoptosis, as indicated by DNA fragmentation, chromatin condensation, and nuclear disintegration. LfcinB killed B-lymphoma cells more efficiently at low serum concentrations and was inhibited in the presence of exogenous bovine serum albumin, suggesting partial neutralization of cationic LfcinB by anionic serum components. LfcinB-induced apoptosis in B-lymphoma cells was caspase-independent since caspase-3 activation was not detected by Western blotting and the general caspase inhibitor z-VAD-fmk did not prevent LfcinB-induced DNA fragmentation. Importantly, immune-deficient SCID/beige mice that were inoculated intravenously with Ramos B-lymphoma cells in order to model B-cell NHL exhibited extended survival following systemic administration of LfcinB, indicating that LfcinB warrants further investigation as a novel therapeutic agent for the possible treatment of B-cell NHL. Copyright 2010 Elsevier Inc. All rights reserved.

  7. 7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells

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    Pereira Paulo

    2004-06-01

    Full Text Available Abstract Background Connexin43 (Cx43 is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols affect gap junction intercellular communication (GJIC. Results Primary cultures of lens epithelial cells (LEC were incubated with 7-ketocholesterol (7-Keto, 25-hydroxycholesterol (25-OH or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake. Conclusions Results demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication

  8. mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

    Science.gov (United States)

    Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

    2016-08-15

    The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation

  9. MicroRNA Expression Profile in Bovine Granulosa Cells of Preovulatory Dominant and Subordinate Follicles during the Late Follicular Phase of the Estrous Cycle.

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    Samuel Gebremedhn

    Full Text Available In bovine, ovarian follicles grow in a wave-like fashion with commonly 2 or 3 follicular waves emerging per estrous cycle. The dominant follicle of the follicular wave which coincides with the LH-surge becomes ovulatory, leaving the subordinate follicles to undergo atresia. These physiological processes are controlled by timely and spatially expressed genes and gene products, which in turn are regulated by post-transcriptional regulators. MicroRNAs, a class of short non-coding RNA molecules, are one of the important posttranscriptional regulators of genes associated with various cellular processes. Here we investigated the expression pattern of miRNAs in granulosa cells of bovine preovulatory dominant and subordinate follicles during the late follicular phase of bovine estrous cycle using Illumina miRNA deep sequencing. In addition to 11 putative novel miRNAs, a total of 315 and 323 known miRNAs were detected in preovulatory dominant and subordinate follicles, respectively. Moreover, in comparison with the subordinate follicles, a total of 64 miRNAs were found to be differentially expressed in preovulatory dominant follicles, of which 34 miRNAs including the miR-132 and miR-183 clusters were significantly enriched, and 30 miRNAs including the miR-17-92 cluster, bta-miR-409a and bta-miR-378 were significantly down regulated in preovulatory dominant follicles. In-silico pathway analysis revealed that canonical pathways related to oncogenesis, cell adhesion, cell proliferation, apoptosis and metabolism were significantly enriched by the predicted target genes of differentially expressed miRNAs. Furthermore, Luciferase reporter assay analysis showed that one of the differentially regulated miRNAs, the miR-183 cluster miRNAs, were validated to target the 3'-UTR of FOXO1 gene. Moreover FOXO1 was highly enriched in granulosa cells of subordinate follicles in comparison with the preovulatory dominant follicles demonstrating reciprocal expression pattern

  10. Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

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    Isabelle Hue

    Full Text Available In addition to nourishing the embryo, extra-embryonic tissues (EETs contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC, endoderm (bXEC, and mesoderm (bXMC cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify "cores" of cell-type-specific (and substrate-independent genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21-D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

  11. A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

    Science.gov (United States)

    Solarte, Víctor Alfonso; Conget, Paulette; Vernot, Jean-Paul; Rosas, Jaiver Eduardo; Rivera, Zuly Jenny; García, Javier Eduardo; Arango-Rodríguez, Martha Ligia

    2017-01-01

    Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-25)4, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25), for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(a)anthracene, and tumors were treated with one of the following peptides: LfcinB(20-25)4, LfcinB(20-25), or vehicle (control). Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-25)4 and LfcinB(20-25) was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively). Also, LfcinB(20-25)4 caused acellularity in the parenchymal tumor compared with LfcinB(20-25) and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-25)4 and LfcinB(20-25) induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively). Moreover, although the lowest inflammatory response was achieved when LfcinB(20-25)4 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25). In addition the cellular damage and selectivity of the LfcinB(20-25)4 peptide was evaluated in vitro. These assays showed that LfcinB(20-25)4 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-25)4 could be considered as a new therapeutic agent for the treatment of OSCC.

  12. A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

    Directory of Open Access Journals (Sweden)

    Víctor Alfonso Solarte

    Full Text Available Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-254, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25, for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(aanthracene, and tumors were treated with one of the following peptides: LfcinB(20-254, LfcinB(20-25, or vehicle (control. Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-254 and LfcinB(20-25 was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively. Also, LfcinB(20-254 caused acellularity in the parenchymal tumor compared with LfcinB(20-25 and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-254 and LfcinB(20-25 induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively. Moreover, although the lowest inflammatory response was achieved when LfcinB(20-254 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25. In addition the cellular damage and selectivity of the LfcinB(20-254 peptide was evaluated in vitro. These assays showed that LfcinB(20-254 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-254 could be considered as a new therapeutic agent for the treatment of OSCC.

  13. Brevenal inhibits pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells.

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    César Mattei

    Full Text Available Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and beta-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

  14. Brevenal inhibits pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells.

    Science.gov (United States)

    Mattei, César; Wen, Peter J; Nguyen-Huu, Truong D; Alvarez, Martha; Benoit, Evelyne; Bourdelais, Andrea J; Lewis, Richard J; Baden, Daniel G; Molgó, Jordi; Meunier, Frédéric A

    2008-01-01

    Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and beta-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

  15. Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

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    Ana Teresa B.F. Antonangelo

    2012-09-01

    Full Text Available Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC. The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

  16. , , , , , and Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid

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    S. H. Choi

    2015-03-01

    Full Text Available We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC and intramuscular preadipocytes (IPA were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS/Dulbecco’s Modified Eagle Medium (DMEM and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC or 5% FBS/DMEM (IPA with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001 carnitine palmitoyltransferase-1 beta (CPT1β gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases. Oleic and linoleic acid decreased (p = 0.001 stearoyl-CoA desaturase (SCD gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

  17. Molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in bovine mammary epithelial cells.

    Science.gov (United States)

    Yu, Cuiping; Luo, Chaochao; Qu, Bo; Khudhair, Nagam; Gu, Xinyu; Zang, Yanli; Wang, Chunmei; Zhang, Na; Li, Qingzhang; Gao, Xuejun

    2014-12-15

    14-3-3γ, an isoform of the 14-3-3 protein family, was proved to be a positive regulator of mTOR pathway. Here, we analyzed the function of 14-3-3γ in protein synthesis using bovine mammary epithelial cells (BMECs). We found that 14-3-3γ interacted with eIF1AX and RPS7 by 14-3-3γ coimmunoprecipitation (CoIP) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) peptide mass fingerprinting analysis. These interactions of 14-3-3γ with eIF1AX and RPS7 were further confirmed by colocalization and fluorescence resonance energy transfer (FRET) analysis. We also found that methionine could promote protein synthesis and trigger the protein expression levels of 14-3-3γ, eIF1AX and RPS7. Analysis of overexpression and inhibition of 14-3-3γ confirmed that it positively affected the protein expression levels of eIF1AX, RPS7, Stat5 and mTOR pathway to promote protein synthesis and cell proliferation in BMECs. We further showed that overexpression of eIF1AX and RPS7 also triggered protein translation and cell proliferation. From these results, we conclude that molecular network including eIF1AX, RPS7, and 14-3-3γ regulates protein translation and cell proliferation in BMECs. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Identification and characterization of phospholipase A2 (PLA2) in bovine pulmonary endothelial cells (BPEC)

    International Nuclear Information System (INIS)

    Martin, T.W.; Wysolmerski, R.B.; Lagunoff, D.

    1986-01-01

    Phosphatidylcholine labeled in the sn-2 position with 3 H-oleic acid was used to measure PLA 2 in cell sonicates (CS) prepared from confluent cultures of BPEC. Substrate at 10-200 μM was incubated with 5-30 μg of CS protein in HEPES buffer at 37 0 C. A plot of 3 H-oleic acid release vs time was linear and proportional to the amount of CS protein. Lineweaver-Burk plots of the data were linear with V/sub max/ = 22.2 nmole/mg protein/hr and K/sub d/ = 121 μM. Under these conditions, phospholipase C activity was 20-fold lower, and phospholipase A 1 activity was not detectable. PLA 2 activity was pH-dependent with optima at 4.5 and 7.5. Ca ++ was not required for activity, and addition of up to 10 mM Ca ++ to CS in EDTA increased activity by only 10-20%. After centrifugation of CS at 100,000 g for 90 min, 62% of the PLA 2 activity was recovered in the particular fraction. Triton X-100 (0.006-0.4%) inhibited PLA 2 up to 90%, whereas 2 mM deoxycholate produced nearly 3-fold activation. Of several agents tested, bromophenacylbromide (BPB) was the most effective inhibitor. Treatment of CS with BPB at 37 0 C for 30 min produced up to 9% inhibition (K/sub i/ = 5 μM). Phenylmethanesulfonyl fluoride at 200 μm produced 41% inhibition. Quinacrine at 1 mM inhibited PLA 2 by 18%. These data define characteristics of BPEC PLA 2 that should prove useful in studies of the role of this enzyme in specific cellular functions

  19. 77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-05-21

    ... Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY: Animal and Plant Health... derived from bovines with regard to bovine spongiform encephalopathy. This action will allow interested... importation of live bovines and products derived from bovines with regard to bovine spongiform encephalopathy...

  20. A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

    Directory of Open Access Journals (Sweden)

    Ustav Mart

    2002-04-01

    Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

  1. Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells.

    Science.gov (United States)

    Kang, Minkyung; Jeong, Wooyoung; Bae, Hyocheol; Lim, Whasun; Bazer, Fuller W; Song, Gwonhwa

    2018-03-01

    Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell-to-cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1-Eph system and mechanisms of its action on bovine mammary epithelial (MAC-T) cells. The in vitro cultured MAC-T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC-T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3 K/AKT and MAPK signaling, and the proliferation-stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress-related proteins and protected MAC-T cells from stress-induced cell death. Finally, ephrin A1 alleviated LPS-induced cell death through down-regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A-ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals. © 2017 Wiley Periodicals, Inc.

  2. Characterization of insulin-like growth factor I and insulin receptors on cultured bovine adrenal fasciculata cells. Role of these peptides on adrenal cell function

    International Nuclear Information System (INIS)

    Penhoat, A.; Chatelain, P.G.; Jaillard, C.; Saez, J.M.

    1988-01-01

    We have characterized insulin-like growth factor I (IGF-I) and insulin receptors in cultured bovine adrenal cells by binding and cross-linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell for IGF-I were 1.4 +/- (SE) 0.3 x 10(-9) M and 19,200 +/- 2,100, respectively. Under reduction conditions, disuccinimidyl suberate cross-linked [ 125 I]iodo-IGF-I to one receptor complex with an Mr of 125,000. Adrenal cells also contain specific insulin receptors with an apparent dissociation constant (Kd) of 10(-9) M. Under reduction conditions [ 125 I]iodo-insulin binds to one band with an approximate Mr of 125,000. IGF-I and insulin at micromolar concentrations, but not at nanomolar concentrations, slightly stimulated DNA synthesis, but markedly potentiated the mitogenic action of fibroblast growth factor. Adrenal cells cultured in a serum-free medium containing transferrin, ascorbic acid, and insulin (5 micrograms/ml) maintained fairly constant angiotensin-II (A-II) receptor concentration per cell and increased cAMP release on response to ACTH and their steroidogenic response to both ACTH and A-II. When the cells were cultured in the same medium without insulin, the number of A-II receptors significantly decreased to 65% and the increased responsiveness was blunted. Treatment of such cells for 3 days with increasing concentrations of IGF-I (1-100 ng/ml) produced a 2- to 3-fold increase in A-II receptors and enhanced the cAMP response (3- to 4-fold) to ACTH and the steroidogenic response (4- to 6-fold) to ACTH and A-II. These effects were time and dose dependent (ED50 approximately equal to 10(-9) M). Insulin at micromolar concentrations produced an effect similar to that of IGF-I, but at nanomolar concentrations the effect was far less

  3. The modulation of the phosphorylation status of NKCC1 in organ cultured bovine lenses: Implications for the regulation of fiber cell and overall lens volume.

    Science.gov (United States)

    Vorontsova, Irene; Donaldson, Paul J; Kong, Zhiying; Wickremesinghe, Chiharu; Lam, Leo; Lim, Julie C

    2017-12-01

    In previous work, we have shown the Sodium/Potassium/2 Chloride Cotransporter (NKCC1) to be a key effector of lens fiber cell volume regulation. Since others have shown that the activity of NKCC1 is regulated via its phosphorylation status, the purpose of this study was to investigate whether NKCC1 phosphorylation can be modulated in organ cultured bovine lenses, and to see how this relates to changes in lens wet weight. Western blotting was first used to confirm the expression of NKCC1, phosphorylated NKCC1 (NKCC1-P) and the regulatory kinases WNK/SPAK and phosphatases PP1/PP2A in bovine lenses at the protein level. Changes to NKCC1-P status were then assessed by organ culturing bovine lenses in either isotonic, hypertonic or hypotonic solutions in the presence or absence of the NKCC inhibitor, bumetanide, or phosphatase inhibitors okadaic acid and calyculin A. After 1-22 h of culturing, lenses were weighed, assessed for transparency and the cortical protein fractions analyzed by western blot using antibodies to detect total NKCC1 and NKCC1-P. NKCC1, NKCC1-P, SPAK, PP1 and PP2A were all detected in the membrane fraction of bovine lenses. Under hypertonic conditions, NKCC1 is phosphorylated and activated to mediate a regulatory volume increase. Finally, NKCC1-P signal increased in the presence of phosphatase inhibitors indicating that PP1/PP2A can dephosphorylate NKCC1. These results show that the phosphorylation status and hence activity of NKCC1 is dynamically regulated and that in response to hypertonic stress, NKCC1 activity is increased to effect a regulatory volume increase that limits cell shrinkage. These findings support the view that the lens dynamically regulates ion fluxes to maintain steady state lens volume, and suggest that dysfunction of this regulation maybe an initiating factor in the localized fiber cell swelling that is a characteristic of diabetic lens cataract. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Bovine leukemia virus infection in cattle of China: Association with reduced milk production and increased somatic cell score.

    Science.gov (United States)

    Yang, Y; Fan, W; Mao, Y; Yang, Z; Lu, G; Zhang, R; Zhang, H; Szeto, C; Wang, C

    2016-05-01

    The main objective of this study was to investigate the individual cow effect of bovine leukemia virus (BLV) infection on milk production and somatic cell score (SCS). The fluorescence resonance energy transfer (FRET) quantitative PCR established in this study and a commercial ELISA kit revealed that 49.1% of dairy cattle (964/1,963) from 6 provinces of China and 1.6% of beef cattle (22/1,390) from 15 provinces were BLV positive. In a detailed study of 105 cows, BLV was found most commonly in buffy coat samples that also had highest copy numbers (10(4.75±1.56) per mL); all cows negative for BLV in buffy coat samples were also negative in vaginal swab, milk, and fecal samples. Copy numbers of BLV were 10(2.90±0.42)/gram of feces, 10(0.83±0.62)/mL of milk, and 10(2.18±0.81) per vaginal swab. The BLV-positive cows had significantly lower milk production in the early (26.8 vs. 30.9kg) and middle stages of lactation (22.2 vs. 26.1kg) in animals with ≥4 parities than the BLV-negative cows; they also had significantly higher SCS in early and middle lactation stages (early=5.2 vs. 4.3; middle=4.9 vs. 3.9) in animals with ≥4 parities. Milk production and SCS did not significantly differ between the BLV-infected and -uninfected cows when they were in the late lactation stage or in animals with ≤3 parities. Taken together, our results indicate that BLV infections are widespread in the dairy farms of China. Vaginal secretions and feces may be involved in BLV transmission. A BLV infection may result in reduced milk yield and increased SCS in a parity and lactation stage-restricted manner. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Spectral cumulus parameterization based on cloud-resolving model

    Science.gov (United States)

    Baba, Yuya

    2018-02-01

    We have developed a spectral cumulus parameterization using a cloud-resolving model. This includes a new parameterization of the entrainment rate which was derived from analysis of the cloud properties obtained from the cloud-resolving model simulation and was valid for both shallow and deep convection. The new scheme was examined in a single-column model experiment and compared with the existing parameterization of Gregory (2001, Q J R Meteorol Soc 127:53-72) (GR scheme). The results showed that the GR scheme simulated more shallow and diluted convection than the new scheme. To further validate the physical performance of the parameterizations, Atmospheric Model Intercomparison Project (AMIP) experiments were performed, and the results were compared with reanalysis data. The new scheme performed better than the GR scheme in terms of mean state and variability of atmospheric circulation, i.e., the new scheme improved positive bias of precipitation in western Pacific region, and improved positive bias of outgoing shortwave radiation over the ocean. The new scheme also simulated better features of convectively coupled equatorial waves and Madden-Julian oscillation. These improvements were found to be derived from the modification of parameterization for the entrainment rate, i.e., the proposed parameterization suppressed excessive increase of entrainment, thus suppressing excessive increase of low-level clouds.

  6. Tripolar acytokinetic mitosis and formation of feto-maternal syncytia in the bovine placentome: different modes of the generation of multinuclear cells.

    Science.gov (United States)

    Klisch, K; Pfarrer, C; Schuler, G; Hoffmann, B; Leiser, R

    1999-08-01

    The vast majority of trophoblast giant cells in the ruminant placenta are binuclear and are believed to derive from mononuclear trophoblastic cells by a single acytokinetic mitosis. There is no satisfactory explanation for the generation of the small proportion of trophoblast giant cells with one, three, or more nuclei. In this light-and electronmicroscopic study of bovine placentomal tissue from the second half of gestation, developmental stages of the trophoblast giant cells are investigated. Large mitotic figures indicate mitotic polyploidization, which is proposed to be due to two subsequent acytokinetic mitoses. Tripolar mitoses offer an explanation for the development of trinucleate trophoblast giant cells. Measurements of nuclear volumes in a series of semithin sections revealed that three size classes of trophoblast giant cells occur. The approximately doubling of nuclear volume between each class is thought to reflect different levels of DNA content that result from polyploidization in this cell type. Although trinuclear feto-maternal hybrid cells are the standard outcome of the fusion of binuclear trophoblast giant cells with uterine epithelial cells, some syncytia with at least five nuclei were observed in the uterine epithelium.

  7. Effects of green tea polyphenols, insulin-like growth factor I and glucose on developmental competence of bovine oocytes

    Directory of Open Access Journals (Sweden)

    Zhengguang Wang

    2012-12-01

    Full Text Available The present study examined the effects of green tea polyphenols (GTP, insulin-like growth factor-I (IGF-I and glucose on oocyte in vitro maturation, subsequent embryo development and blastocyst quality in bovine. Cumulus-oocyte complexes (COC were aspirated from the ovaries and cultured in synthetic oviduct fluid supplemented with MEM amino acids (SOFaa media supplemented with one of the following supplements: GTP (0, 10, 15 and 20 µM, IGF-I (0, 50, 100 and 150 ng/mL or glucose (0, 1.5, 5.6 and 20 mM for 24 h. The results showed that oocytes cultured in media supplemented with 15 µM GTP, 100 ng/mL IGF-I and 5.6 mM glucose, in separate experiments, have higher cleavage and blastocyst rates compared with oocytes cultured in media without or with other concentration of GTP, IGF-I and glucose. Then these three substances with the concentration above were added together into SOFaa media and constituted a modified medium (Modified SOFaa. The COC were cultured in control SOFaa media and modified SOFaa media, respectively. The results showed that modified SOFaa media increased the intracellular glutathione concentration of matured oocytes, blastocyst rates and total cell numbers and cell numbers of inner cell mass per blastocyst compared with the control. Supplementing of GTP, IGF-I and glucose synchronously to maturation media can increase the intracellular GSH concentration of oocytes after in vitro maturation, and improve the embryo development and blastocyst quality in bovine.

  8. Bovine Tuberculosis, A Zoonotic Disease

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    Tarmudji

    2008-12-01

    Full Text Available Bovine tuberculosis is caused by the infection of Mycobacterium tuberculosis var. bovis (M. bovis. This species is one of Mycobacterium tuberculosis complex, can infect wide range of hosts: cattle and other domesticated animals, wild mammals and humans (zoonotic. M. bovis bacterium from infected hosts can be transmitted to other susceptible animals and humans through respiratory excretes and secretion materials. Humans can be infected with M. bovis by ingested M. bovis contaminated animal products, unpasteurised milk from tuberculosis cows or through respiratory route of contaminated aerosol. Bovine tuberculosis at the first stage does not show any clinical sign but as the disease progress in the next stage which may take several months or years, clinical signs may arise, suh as: fluctuative body temperature, anorexia, lost body weight, coughing, oedema of lymph nodes, increased respiratory frequencies. Pathological lesion of bovine tuberculosis is characterised by the formation of granulomas (tubercles, in which bacterial cells have been localised, most in lymph nodes and pulmonum, but can occur in other organs. The granulomas usually arise in small nodules or tubercles appear yellowish either caseus, caseo-calcareus or calcified. In Indonesia, bovine tuberculosis occurred in dairy cattle since 1905 through the imported dairy cows from Holland and Australian. It was unfortunate that until recently, there were not many research and surveilances of bovine tuberculosis conducted in this country, so the distribution of bovine tuberculosis is unknown. Early serological diagnosis can be done on live cattle by means of tuberculin tests under field conditions. Confirmation can be done by isolation and identification of excreted and secreted samples from the slaughter house. Antibiotic treatment and vaccination were uneffective, therefore the effective control of bovine tuberculosis is suggested by tuberculin tests and by slaughtering the selected

  9. Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

    Science.gov (United States)

    Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

    2016-08-01

    Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Identification of bovine leukemia virus tax function associated with host cell transcription, signaling, stress response and immune response pathway by microarray-based gene expression analysis

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    Arainga Mariluz

    2012-03-01

    Full Text Available Abstract Background Bovine leukemia virus (BLV is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type I. The Tax protein of BLV is a transcriptional activator of viral replication and a key contributor to oncogenic potential. We previously identified interesting mutant forms of Tax with elevated (TaxD247G or reduced (TaxS240P transactivation effects on BLV replication and propagation. However, the effects of these mutations on functions other than transcriptional activation are unknown. In this study, to identify genes that play a role in the cascade of signal events regulated by wild-type and mutant Tax proteins, we used a large-scale host cell gene-profiling approach. Results Using a microarray containing approximately 18,400 human mRNA transcripts, we found several alterations after the expression of Tax proteins in genes involved in many cellular functions such as transcription, signal transduction, cell growth, apoptosis, stress response, and immune response, indicating that Tax protein has multiple biological effects on various cellular environments. We also found that TaxD247G strongly regulated more genes involved in transcription, signal transduction, and cell growth functions, contrary to TaxS240P, which regulated fewer genes. In addition, the expression of genes related to stress response significantly increased in the presence of TaxS240P as compared to wild-type Tax and TaxD247G. By contrast, the largest group of downregulated genes was related to immune response, and the majority of these genes belonged to the interferon family. However, no significant difference in the expression level of downregulated genes was observed among the Tax proteins. Finally, the expression of important cellular factors obtained from the human microarray results were validated at the RNA and protein levels by real-time quantitative reverse transcription-polymerase chain reaction and western blotting

  11. Human Serum is as Efficient as Fetal Bovine Serum in Supporting Proliferation and Differentiation of Human Multipotent Stromal (Mesenchymal) Stem Cells In Vitro and In Vivo

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Haack-Sørensen, Mandana; Al-Nbaheen, May

    2011-01-01

    BACKGROUND: Human multipotent stromal (skeletal, mesenchymal) stem cells (hMSC) are employed in an increasing number of clinical trials for tissue regeneration of age-related degenerative diseases. However, routine use of fetal bovine sera (FBS) for their in vitro expansion is not optimal and may......) or adipocytic markers (PPAR-gamma2, lipoprotein lipase (LPL), aP2), respectively. In order to test for the functional capacity of hMSC-TERT that have been maintained in long-term cultures in the presence of HuS vs. FBS, the cells were mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and implanted...... subcutaneously in immune deficient mice. hMSC maintained in HuS vs. FBS formed comparable heterotopic bone. DISCUSSION: Human serum can support proliferation and differentiation of hMSC in vitro and can maintain their bone forming capacity in vivo. The use of human serum in cell cultures of hMSC intended...

  12. Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

    DEFF Research Database (Denmark)

    Holm, Peter; Shukri, Naseer Mahmoud; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

  13. Proteolytic Activity of the 26S Proteasome is required for the Meiotic Resumption, Germinal Vesicle Breakdown and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes Matured In Vitro

    Czech Academy of Sciences Publication Activity Database

    Yi, Y. J.; Nagyová, Eva; Manandhar, G.; Procházka, Radek; Šutovsky, M.; Park, C. S.; Šutovský, P.

    2008-01-01

    Roč. 78, - (2008), s. 115-126 ISSN 0006-3363 R&D Projects: GA ČR GA305/05/0960 Institutional research plan: CEZ:AV0Z50450515 Keywords : cumulus expansion * FSH * germinal vesicle breakdown Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.469, year: 2008

  14. Proteasomal activity has multiple functions in oocyte meiosis, in cumulus expansion, in synthesis and processing of cumulus extracellular matrix and steroidogenesis

    Czech Academy of Sciences Publication Activity Database

    Nagyová, Eva

    2014-01-01

    Roč. 3, č. 2 (2014), s. 163-163 ISSN 2161-1017. [International Conference on Endocrinology /2./. 20.10.2014-22.10.2014, Chicago] Institutional support: RVO:67985904 Keywords : oocyte-cumulus complexes Subject RIV: EB - Genetics ; Molecular Biology

  15. PRODUCTION OF EMBBRYONIC STEM CELLS FROM INNER CELL MASS OF BLASTOCYST ISOLATED BY ENZYMATIC AND IMMUNOSURGERY METHODS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2008-03-01

    Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

  16. Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer

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    Carolina Gonzales da Silva

    Full Text Available ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs that had not yet been tested for bovine embryo production by nuclear transfer (NT. Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+ were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%. Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

  17. Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

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    Kun Taek Park

    Full Text Available Phylogenic comparisons of the mononuclear phagocyte system (MPS of humans and mice demonstrate phenotypic divergence of dendritic cell (DC subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2, plasmacytoid DC (pDC, and monocyte derived DC (MoDC. DC of Artiodactyla (pigs and ruminants can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC. Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ. Functional analysis revealed each of these populations can take up and process antigens (Ags, present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

  18. TNAP and EHD1 are over-expressed in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties.

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    Barbara Deracinois

    Full Text Available Although the physiological properties of the blood-brain barrier (BBB are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established in vitro BBB model developed in our laboratory, we performed a differential nano-LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial