Cytogenetic analysis of lymphoid blood cells in bovine leucosis]

For the cytogenetic analysis lymphocytes of the peripheral blood were used that had been obtained from cows suffering from leucosis. The blood was taken from a diseased cow, from its 15 months old daughter suffering from leucosis, and from the healthy bull-father (NAT-47). The diagnosis of leucosis was determined by means of hematological examination. In the cow 139 metaphase plates were evaluated, in the daughter 118, and in the bull 132. On the one hand, normoploidy was determined and on the other hand, chromosome aberrations. In the cow 31.0 p. c. of chromosome aberrations were found, in the daughter 32.3 p. c., and in the bull 37.2 p. c. Breaks in X chromosomes were found in the cow (6.7 p. c.) and in the daughter (1.7 p. c.). Longitudinal diversion of arms in the centromere in X chromosomes in the vertical axis into two separate arms was found in the cow amounting to 6.5 p. c., in the daughter to 5.9 p. c., and in the bull to only 0.8 p. c.     

牛源犬新孢子虫MAG1基因的克隆及在Vero细胞中表达

为了解牛源犬新孢子虫MAG1基因的生物学特性,本实验应用PCR技术扩增牛源犬新孢子虫MAG1基因,构建真核表达重组质粒pVAX-MAG1,将鉴定正确的pVAX-MAG1重组质粒转染Vero细胞,应用间接荧光检测方法(1FA)和western blot技术检测MAG1基因在Vero细胞中的表达.结果显示,扩增的牛源犬新孢子虫MAG1基因长度为1047bp,与GenBank中登录的MAG1 (EF580924.1)核苷酸序列同源性为99％,IFA检测MAG1基因在Vero细胞中获得瞬时表达,western blot分析表达蛋白的分子量为39 ku,具有较好的反应原性.本实验为新孢子虫病核酸疫苗与诊断试剂盒的研究奠定了基础.     

Growth activity of bovid herpesvirus 1 in bovine follicular oocytes with cumulus cells.

Bovine follicular oocytes collected from bovine ovaries were exposed to bovid herpesvirus 1 (BHV-1). After washings, these oocytes were cultured to mature. As a result BHV-1 could not be removed from the oocytes and could replicate in the oocytes with cumulus cells, but not in the oocytes without the cells. Moreover, the specific fluorescence for BHV-1 was detected in the cumulus cells by a indirect immunofluorescent technique. Therefore these findings suggested BHV-1 could be absorbed in the oocytes but the replication of BHV-1 was done in the cumulus cells.     

Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression

Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.     

Neuroendocrine Regulation of Growth Hormone and Luteinizing Hormone in Fetal and Neonatal Pig

Ontogeny of the production and the regulation of growth hormone and Luteinizing hormone is studied in a series of experiments utilizing male and female pigs at different fetal and neonatal stages. Growth hormone mRNA is detectable in both sexes as early as d. 50 p.c. The mRNA levels increase to reach the maximum levels at d. 95–110 p.c. Plasma levels of GH follow the developmental patterns of GH mRNA. A sex difference is evident around d. 80–90 p.c. with males having higher GH levels than females. The stimulatory but not the inhibitory mechanisms of GH secretion are fully functioning in the pig fetus. LHβ mRNA is detectable earlier in females (d. 50 p.c.) than in males (d. 65 p.c.). Plasma concentrations of LH increase with fetal age in female fetuses, but in male fetuses there is no distinct developmental pattern evident. Basal LH secretion achieves maximum levels in both sexes after birth. Opioids do modulate fetal LH secretion, however, the mode of their action is age-dependent.     

Growth hormone, insulin and glucose concentrations in bovine fetal and maternal plasmas at several stages of gestation

For cows on d 137 (n = 6), 180 (n = 8), 226 (n = 9) and 250 (n = 5) of gestation (Exp. 1), concentrations of insulin and glucose were two- to three-fold less (P less than .01) in fetal venous plasma than in uterine arterial plasma. Concentrations of growth hormone, conversely, were 10- to 20-fold greater (P less than .01) in fetal venous than in uterine arterial plasma. Concentrations of insulin and glucose in maternal and fetal plasmas and concentrations of growth hormone in maternal plasma did not vary with stage of gestation. Concentrations of growth hormone in fetal venous plasma, however, were greater on d 226 and 250 than on d 137 and 180. For cows (n = 6) on d 198 of gestation (Exp. 2), concentrations of insulin and glucose in maternal and fetal plasmas and of growth hormone in maternal plasma remained relatively constant in samples collected every 30 min for 3 h. In contrast, growth hormone concentrations in fetal venous plasma were highly variable and appeared to be episodic, with pulses of 10 to 60 ng/ml in amplitude. No significant correlations were found among concentrations of insulin, glucose and growth hormone in fetal venous plasma. When samples were collected every 15 min for 4 h from cows (n = 5) on d 198 of gestation (Exp. 3), episodes of growth hormone in fetal venous plasma were irregular in amplitude and frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

子宫内膜炎患牛子宫内膜PGR和ESR-1 mRNA的表达特征

为探讨卵巢激素及其子宫内膜受体在奶牛子宫内膜炎中的作用,以产后6～10d健康及患急性化脓性子宫内膜炎的中国荷斯坦奶牛为研究对象,通过ELISA法检测子宫内膜中PGF2a,血清中孕酮和雌二醇浓度,SYBRGreenⅠ荧光定量PCR检测子宫内膜PGR和ESR-1mRNA的表达。结果显示,病牛子宫内膜PGF2α水平为（6.503±0.122）μg/L,血清孕酮为（0.816±0.281）μg/L,血清雌二醇为（35.462±2.523）μg/L,而对照组分别为（14.408±0.285）,（0.543±0.142）,（37.6±1.317）μg/L;子宫内膜炎患牛组PGF2a水平极显著低于对照组（P〈0.01）,孕酮含量显著高于对照组（P〈0.05）,PGF2a与孕酮水平呈显著负相关（r=0.893）,雌二醇含量组间差异不显著。PGR基因的mRNA水平极显著高于对照组（P〈0.01）,而ESR-1基因的mRNA水平低于对照组,且差异极显著（P〈0.01）。上述结果表明,孕酮浓度显著升高及PGR mRNA高表达,可能抑制子宫收缩;ESR-1mRNA低表达使子宫新陈代谢减慢,子宫内膜合成PGF2a功能下降,这些改变有可能促进了产后子宫内膜炎的发生发展。     

Regulation of growth hormone expression by thyrotropin-releasing hormone through the pituitary-specific transcription factor Pit-1 in chicken pituitary

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone beta- (TSHbeta) encoding genes. Pit-1 has been identified in several mammalian and avian species. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and it stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully developed a competitive RT-PCR for the detection of Pit-1 expression in the chicken pituitary, that was sensitive enough to detect picogram levels of Pit-1 mRNA. Applying this method, the effect of TRH injections on Pit-1 mRNA expression was determined in the pituitary of chick embryos and growing chicks. In both 18-day-old embryos and 10-day-old male chicks the Pit-1 mRNA expression was significantly increased following TRH injection, thereby indicating that the stimulatory effects of TRH on several pituitary hormones is mediated via its effect on Pit-1 expression. Therefore, a semi-quantitative RT-PCR method was used to detect possible changes in GH levels. TRH affected the GH mRNA levels at both developmental stages. These results, combined with the data on Pit-1 mRNA expression, indicate that Pit-1 has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH.     

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.     

猪生长激素的研究进展

猪生长激素(porcinegrowthhormone,pGH)是一种由猪脑垂体前叶嗜酸性细胞分泌的单一肽链蛋白质类激素。1920年,Evans和Simpson给大鼠注射牛垂体提取物,首次证实GH具有促生长作用,因此称之为生长激素。在生长轴中,GH是调控动物生长发育的核心,是一种具有广泛生理功能的生长调     

牛Mx1基因重组慢病毒载体的构建及沉默Mx1细胞的获得

采用RT-PCR技术从牛肾细胞中扩增出Mx1基因,构建牛Mx1基因过表达重组载体;针对牛Mx1mRNA序列设计shRNA引物对,构建针对牛Mx1的RNAi载体。将牛Mx1的过表达载体和RNAi载体共转染293T细胞,利用Western blot方法检测牛Mx1蛋白的表达情况,筛选有效沉默牛Mx1的shRNA;将有效沉默Mx1的RNAi载体转染牛胎儿气管原代上皮细胞,经流式细胞分选,Western blot检测,获得沉默牛Mx1的稳定细胞系。结果表明,成功构建了牛Mx1过表达重组载体pcDNA3.1-Mx1和RNAi载体RNAi-Mx1A、RNAi-Mx1B、RNAi-Mx1C,筛选到RNAi-Mx1A沉默牛Mx1效果最好;经流式细胞分选和Western blot检测,获得有效沉默牛Mx1的牛胎儿气管原代上皮细胞,为进一步探讨牛Mx1抗病毒作用及机制的研究奠定试验基础。     

Inhibitors of bovine parvovirus,coronavirus and rotavirus in precolostral and fetal bovine sera

Eleven, 11 and 2 of 11 precolostral sera from normal calves and 13, 14 and 9 of 14 sera from normal fetuses, 7 to 10 months of gestation, neutralized bovine parvovirus, coronavirus and rotavirus, respectively.When assayed by single radial immunodiffusion, all the sera contained IgG at 360 to 1400 mg/dl, and some of them had much smaller amounts of IgM or IgA. Most of the neutralizing activities against bovine coronavirus and rotavirus were readily inactivated by treatment with acetone or 2-mercaptoethanol. Some sera fractionated by Sephadex G-200 gel filtration or starch block electrophoresis had neutralizing activities against bovine parvovirus or coronavirus in fractions containing no detectable amounts of immunoglobulins. These observations seem to indicate the presence of substance(s), other than immunoglobulins, capable of inhibiting replication of bovine parvovirus, coronavirus or rotavirus. The chemical nature and the mode of action of the inhibitors await elucidation.     

Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes

Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.