囊泡相关蛋白——扫尾负鼠中新发现的一种卵母细胞蛋白

在扫尾负鼠卵母细胞中新发现了一种蛋白,即囊泡相关蛋白（VAP1）。该蛋白基因在卵巢或发育的卵母细胞中有表达,与卵母细胞中极性的囊泡形成有关。囊泡相关蛋白在N端的T13～K114区域有与半胱氨酸酶抑制蛋白C相似的结构域,可能具有抑制半胱氨酸酶对蛋白的酶解作用,促进卵母细胞中母源蛋白的积累,调节受精后胚胎的发育。     

猪轮状病毒重组VP7蛋白抗原间接ELISA诊断方法的建立

本研究以纯化的原核表达的猪轮状病毒VP7抗原表位区域为抗原,建立了检测猪轮状病毒抗体的间接ELISA诊断方法。特异性试验表明,该抗原与其他7种常见猪病病毒（TGEV、PEDV、CSFV、PCV2、PRRSV、PPV、PrV）的阳性血清不发生交叉反应,批内和批间重复性试验的变异系数均小于10％;对来自不同猪场的血清的检测结果表明,该ELISA方法与中和试验检测结果符合率达94．8％。本试验建立的ELISA诊断方法具有良好的重复性、敏感性和特异性,为PRV的快速诊断、免疫猪群抗体监测和轮状病毒流行病学调查提供了一种快速、简便的血清学诊断方法。     

H5N1 亚型禽流感病毒NS1的克隆和原核表达

利用RT-PCR技术扩增了一株H5N1亚型禽流感病毒(AIV)的NS1基因,克隆到pGEM T-easy载体上,经序列测定和分析正确后,将该基因插入到pET-32a载体中构建原核表达载体pET-NS1,阳性重组质粒转化BL21感受态细胞,用1mmol/L的IPTG诱导和SDS-PAGE电泳后获得了分子质量约为51ku的NS1融合蛋白。通过Western-blotting发现NS1蛋白可与H5N1亚型AIV单因子血清反应。为建立H5N1亚型AIV野毒株和疫苗株的特异性鉴别方法奠定了基础。     

猪链球菌2型38 000蛋白主要功能区基因的克隆与表达

根据GenBank中已发表的猪链球菌2型38000蛋白基因核苷酸序列,设计合成1对特异性引物,采用PCR方法,以四川分离株猪链球菌2型基因组DNA为模板扩增38000蛋白主要功能区基因。将PCR产物纯化后与pMD18-T连接转化宿主菌DH5α,提取阳性质粒,进行PCR和酶切鉴定。将目的片段定向克隆到表达载体pET32a中,经测序正确后,重组质粒转化入大肠杆菌BL21(DE3),于37℃、0.8mmol/LIPTG条件下进行诱导表达,结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子质量约为35000的重组蛋白,表达产物经纯化后,免疫印迹法(Western blotting)证实该重组蛋白可以与猪链球菌2型阳性血清发生特异性反应。     

牛杀菌／通透性增加蛋白氮端基因的克隆和序列分析

本试验应用RT-PCR技术,参照Genbank报道的序列,从荷斯坦牛中性粒细胞mRNA中扩增出杀菌／通透性增加蛋白（BPI）氮端基因（713bp）,并与pGEM-T-easy载体连接,构建基因重组体pGEM-T-easy-BPI,进行序列测定。结果表明获得长度为713bp的BPI氮端基因。序列分析证实该片段与安哥斯牛BPI氮端基因相比有1个点突变,为同义突变。该基因的克隆为进一步表达该基因奠定了基础。     

伊犁绢蒿体内可塑性营养物质动态变化及分配特征的研究(简报)

2006年3-11月,在新疆阿什里乡对新疆蒿类荒漠草地建群种伊犁绢蒿可溶性碳水化合物、蔗糖、淀粉和粗蛋白进行动态监测。结果表明,伊犁绢蒿中粗蛋白和蔗糖含量居前2位,淀粉含量低,还原糖仅在茎中存在。5月4日-11月14日,可溶性碳水化合物、蔗糖含量呈"升高-降低-再升高-再降低"特征,叶片粗蛋白和淀粉均呈"单峰"曲线,最高为234.05和3.55 mg/g;根粗蛋白基本不变,为67.26 mg/g,茎中呈下降趋势。可塑性营养物质均在10月17日前完成根内贮存。不同生长阶段伊犁绢蒿营养物质在根、茎、叶上分配差异较大。     

Involvement of the ERK1/2 MAPK pathway in insulin-induced S6K1 activation in avian cells

In mammals, insulin regulates S6K1, a key enzyme involved in the control of protein synthesis, via the well-documented phosphoinositide-3'kinase (PI3K) pathway. Conversely, S6K1 is activated by insulin in avian muscle despite the relative insulin insensitivity of the PI3K pathway in this tissue. Mitogen-activated protein kinase (MAPK) cascade is another insulin sensitive pathway. The aim of this study was to explore the potential involvement of the ERK1/2 MAPK pathway in the control of p70 S6 kinase (S6K1) in avian species. Firstly, we characterized ERK1/2 MAPK in various chicken tissues. ERK2 was the only isoform detected in avian species whatever the tissue studied. We also showed that ERK2 is activated in vivo by insulin in chicken muscle. The regulation and the role of ERK2 in insulin signaling were next investigated in chicken hepatoma cells (LMH) and primary myoblasts. Insulin stimulation led to ERK2 and S6K1 phosphorylation, and concomitantly increased kinase activity. U0126, an inhibitor of the ERK MAPK pathway, completely abolished insulin-induced S6K1 phosphorylation and activity in chicken myoblasts, whereas its effect was only partial in LMH cells. In conclusion, these results show that ERK1/2 MAPK is involved in the control of S6K1 by insulin in chicken cells, particularly myoblasts.     

Effects of ghrelin and its analogues on chicken ovarian granulosa cells

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1-28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1-18 and 1-5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P(4)), testosterone (T), estradiol (E(2)) and arginine-vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1-28, GHRP6 and ghrelin 1-18, but not by ghrelin 1-5. Expression of bcl-2 was suppressed by addition of ghrelin 1-28, GHRP6 and ghrelin 1-5, but promoted by ghrelin 1-18. The occurrence of PCNA was reduced by ghrelin 1-28, GHRP6, ghrelin 1-18 and ghrelin 1-5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1-28, GHRP6 and ghrelin 1-18, but not ghrelin 1-5. The accumulation of PKA decreased after treatment with ghrelin 1-28 and increased after treatment with GHRP6 and ghrelin 1-18 but not ghrelin 1-5. Secretion of P(4) by ovarian follicular fragments was decreased after addition of ghrelin 1-28 or ghrelin 1-5 but stimulated by GHRP6 and ghrelin 1-18. Testosterone secretion was inhibited by ghrelins 1-28 and 1-18, but not by GHRP6 or ghrelin 1-5. Estradiol secretion was reduced after treatment with ghrelin 1-28 but stimulated by ghrelins 1-18 and 1-5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1-28, GHRP6 and ghrelin 1-18, but inhibited by ghrelin 1-5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions-apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5-18 may be responsible for the major effects of ghrelin on the avian ovary.     

Estimation of protein requirement for maintenance in adult parrots (Amazona spp.) by determining inevitable N losses in excreta

Especially in older pet birds, an unnecessary overconsumption of protein--presumably occurring in human custody--should be avoided in view of a potential decrease in the excretory organs' (liver, kidney) efficiency. Inevitable nitrogen (N)-losses enable the estimation of protein requirement for maintenance, because these losses have at least to be replaced to maintain N equilibrium. To determine the inevitable N losses in excreta of adult amazons (Amazona spp.), a frugivor-granivorous avian species from South America, adult amazons (n = 8) were fed a synthetic nearly N-free diet (in dry matter; DM: 37.8% starch, 26.6% sugar, 11.0% fat) for 9 days. Throughout the trial, feed and water intake were recorded, the amounts of excreta were measured and analysed for DM and ash content, N (Dumas analysis) and uric acid (enzymatic-photometric analysis) content. Effects of the N-free diet on body weight (BW) and protein-related blood parameters were quantified and compared with data collected during a previous 4-day period in which a commercial seed mixture was offered to the birds. After feeding an almost N-free diet for 9 days, under the conditions of a DM intake (20.1 g DM/bird/day) as in seeds and digestibility of organic matter comparable with those when fed seeds (82% and 76% respectively), it was possible to quantify the inevitable N losses via excrements to be 87.2 mg/bird/day or 172.5 mg/kg BW(0.75)/day. Assuming a utilization coefficient of 0.57 this leads to an estimated protein need of approximately 1.9 g/kg BW(0.75)/day (this value does not consider further N losses via feathers and desquamated cells; with the prerequisite that there is a balanced amino acid pattern).     

Composition of individual nucleobases in diets containing different products from bacterial biomass grown on natural gas, and digestibility in mink (Mustela vison)

The objectives of the present study were to quantify the amount of nucleic acids, to examine the nucleobase composition and to determine the digestibility of individual nucleobases in diets containing various products from bacterial biomass grown on natural gas, including autolytic and hydrolytic fractions, using mink (Mustela vison) as a model animal. The diets consisted of cod fillet (control), commercial basic BioProtein, and five experimentally produced autolytic and hydrolytic fractions of the bacterial protein meal as protein sources. Each diet was assigned to four adult male mink, housed individually in cages equipped for controlled feeding and quantitative collection of faeces. Faeces were collected for 4 days, and the individual nucleobases in diet and faeces were analysed by HPLC after hydrolysis using HClO(4). The content of nucleobases in the diets containing bacterial protein meal was 37% to 205% higher than in the control diet. The nucleobase-nitrogen in the control diet amounted to 4.3% of the total nitrogen content, whereas the experimental bacterial protein diets contained from 7.4% to 17.4% of the total N content in the form of nucleic acids. The various methods used to produce the bacterial protein fractions clearly affected both the amount of nucleic acids and the molar proportions of the individual nucleobases. The average digestibility of the nucleobases was 95%, and all the individual nucleobases were highly digestible. Uracil showed the highest digestibility (on average 96.8%), whereas thymine showed the lowest digestibility (on average 93.6%).