半胱胺对藏绵羊断奶羔羊瘤胃pH值、总脱氢酶及TVFA的影响

试验研究了半胱胺(Cs)对藏绵羊断奶羔羊瘤胃pH值、总脱氢酶及TVFA的影响。将10只(公母各半)80日龄左右、体重平均为(16.00±0.31)kg、装有永久性瘤胃瘘管的藏绵羊断奶羔羊随机分为2组(试验组和对照组),在日粮精料中添加300 mg/(kg·BW)的Cs,隔日添加1次,试验期42 d。正式试验当日及试验第7、14、21、28、35天的7:00、9:00、11:00、13:00、15:00、17:00、19:00、21:00、23:00、1:00、3:00、5:00于颈静脉采血样10 mL,同时采集瘤胃液10 mL,测定瘤胃pH值、总脱氢酶的活性和TVFA。检测结果表明,试验组精料中添加Cs使藏绵羊断奶羔羊瘤胃液pH值升高;血清尿素氮浓度降低,与对照组相比差异极显著(P0.01);瘤胃液总脱氢酶活力提高极其明显(P0.01);整个试验期内,半胱胺对瘤胃TVFA的影响极显著(P0.01)。     

Acquisition of the potential for sperm motility in steelhead (Oncorhynchus mykiss): effect of pH on dynein ATPase

Salmonid sperm pre-incubated at extracellular pH (pH_e) values less than about 7.4 do not become motile upon water activation whereas sperm maintained above about pH 8.0 demonstrate maximal motility upon activation. The basis for this permissive effect of elevated pH_e on sperm motility is not known. Since it is conceivable that the pH sensitivity of dyneinATPase (the molecular motor that drives flagellar movement) could be the basis of, or contribute to this pH dependency, the pH sensitivity of this enzymatic activity was evaluated in membrane-permeabilized axonemes (isolated flagella) ofsteelhead sperm. DyneinATPase activity was found to be sensitive to pH. This activity in permeabilized axonemes was about 3.5-fold higher at pH 7.6 compared to 7.0. To determine whether the pH sensitivity ofATP regeneration might affect the interpretation of the effect of pH on dyneinATPase activity, the pH sensitivities of creatine kinase and adenylate kinase were established. The rates ofATP generation by these enzymes were insensitive to pH between 6.5 and 8.0. The results of these studies are consistent with the hypothesis that prior maintenance at pH_e, in part, controls the potential for sperm motility upon water activation via an influence on dyneinATPase activity. However, the potential for motility ofsteelhead sperm is particularly sensitive to prior maintenance at pH_e values between about 7.4 and 8.0 whereas the dyneinATPase activity of permeabilized axonemes was particularly sensitive to pH values between 7.0 and 7.6. Phosphorus NMR spectroscopy was used to determine that sperm intracellular pH (pH_i) increased with increasing pH_e between 7.0 and 8.5 and pH_i was, on average 0.4–0.5 pH units lower than pH_e. Therefore the pH_e sensitivity of the potential for motility appears to correspond to the pH_i sensitivity of dyneinATPase activity. The data indicate that pH_i is directly related to pH_e and that prior incubation at pH_e may, in part, control the sperm's potential for motility upon water activation via an influence on dyneinATPase activity.     

Water quality affects SDH activity, protein content and RNA:DNA ratios in fish (Catla catla, Labeo rohita and Oreochromis mossambicus) raised in ponds of a sewage-fed fish farm

The spatial and seasonal variability of the respiratory enzyme succinate dehydrogenase and the protein content were examined in different tissues of fish cultured in three ponds along the effluent gradient of a sewage-fed fish farm. Indian major carp Catla catla (150-230 g) and Labeo rohita (60-190 g) cultured in both the middle and last points of the sewage effluent (stocking pond 1) and (stocking pond 4) and Oreochromis mossambicus (50-160 g), a naturally growing fish of the inlet (facultative pond) and the out let of the sewage effluent (stocking pond 4) were procured every month during the period of January-December, 2005 and were subjected to determination of succinate dehydrogenase activity, total protein, DNA and RNA contents from gill, liver and muscle tissue respectively. The SDH activity of all three test fishes (Catla catla, Labeo rohita and Oreochromis mossambicus) was reduced significantly (ANOVA; P < 0.05) when cultured in SP-4 compared to SP-1 in the case of Catla catla and Labeo rohita and in facultative pond in the case of Oreochromis mossambicus.Conspicuous differences in the SDH activity of fish between the last and first stocking pond or the facultative pond were clearly due to the result of the differences in water quality. There was a direct relationship between SDH activity in gill tissue of any of the fish investigated and ammonia-N concentration of water or water pH. This shows that the respiratory activity of these fishes was strongly affected by the ammonia and pH of water. In other words, this suggests that as the distance from the point source increases, there was a substantial improvement of water quality in the ponds located along the sewage effluent gradient. Evidently, there is a progressive pattern of growth, survival and physiological health of fish and abundance of favorable diversity of food organisms with rich biodiversity.     

Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout (Oncorhynchus mykiss). II. Adaptive response of glucose 6-phosphate dehydrogenase activity to high-carbohydrate/low-protein and high-fat/non-carbohydrate diets

The influence of long-term administration of high-carbohydrate/low-protein and high-fat/non-carbohydrate diets were studied in relation to kinetic behaviour of glucose 6-phosphate dehydrogenase in liver and kidney of rainbow trout. In all cases studied, the saturation curves of these enzyme showed typical hyperbolic kinetics without evidence of sigmoidicity. After 30 days of feeding with a high-fat diet (170 g kg^?1), there was a significant decrease in V_max and specific activity (45%) as well as catalytic efficiency (39%) without changes in K_m or activity ratio of hepatic glucose 6-phosphate dehydrogenase. These changes agree more with a clearly decreased cell concentration than with an inhibition of the pre-existing enzyme. The administration of a high-carbohydrate diet (60 g kg^?1), contrary to what was previously thought, decreased V_max by 21% and specific activity and catalytic efficiency by 30%, without significant changes in the other kinetic parameters of the hepatic enzyme. The kinetic behaviour under these nutritional conditons was due to the rejection of this diet by the fish and thus could be considered a low-feeding situation. On the other hand, no variations in the kinetics of renal glucose 6-phosphate dehydrogenase were found, clearly demonstrating that in this organ, the pentosephosphate pathway showed no adaptive response related to fattyacid and other lipid synthesis. The activity of the renal enzyme was consistently half that of the hepatic enzyme.     

大豆脂肪酸脱氢酶GmFAD3C-1基因的克隆及功能分析

研究通过对大豆脂肪酸脱氢酶GmFAD3家族中4个关键酶基因序列进行比对,与对照JN18相比,大豆低亚麻酸突变体MT72中GmFAD3C-1基因在起始密码子后+966bp处存在一个碱基位点的缺失（G→?）,产生移码突变,导致氨基酸序列上产生较大变化（该突变基因命名为gmfad3c-1）。构建GmFAD3C-1基因超表达及CRISPR/Cas9编辑载体,利用花粉管通道法获得T₂转化植株。脂肪酸脱氢酶酶活测定结果显示,超表达植株T₂籽粒中,酶活与对照相比上升47.62%~78.85%,编辑载体T₂籽粒中,酶活与对照相比下降25.67%~47.11%。脂肪酸相对含量测定的结果进一步表明,GmFAD3C-1基因的表达与植株亚麻酸含量密切相关。     

蛋白质磷酸化在油菜素内酯信号通路中的作用

油菜素内酯是植物体内一类重要的类固醇激素,参与调控植物生长发育过程。对拟南芥的大量研究基本阐明了BR信号转导网络中的主要通路。在BR信号转导过程中,蛋白质可逆磷酸化参与了多个重要环节的调控。该文综述了植物体内BR信号转导过程中蛋白质磷酸化的作用。     

猪链球菌丝氨酸/苏氨酸激酶基因缺失株的构建及其致病性

为了研究丝氨酸/苏氨酸磷激酶(STK)在猪链球菌致病过程中的作用,利用大肠杆菌-猪链球菌穿梭质粒p SET4s构建STK基因缺失株△stk。结果显示,stk基因缺失后,缺失株△stk易形成长链状结构;缺失株的半致死剂量(LD50)较亲本株高7倍,对CD1小鼠的致病能力降低;体内定植试验结果表明缺失株△stk在小鼠体内各器官的定植能力显著降低。这些数据表明STK与猪链球菌2型(SS2)的致病性相关。     

泡桐丛枝植原体胸苷酸激酶的原核表达、纯化及酶活性测定

胸苷酸激酶是d TTP从头合成和补救途径的关键酶,催化d TMP形成d TDP,在DNA复制和生物的生存中发挥着必不可少的作用。本文在前期研究的基础上,对从泡桐丛枝(Pa WB)植原体中获得的的3个同源蛋白TMK-a-1、TMK-a-2及TMK-b与已报道的小麦蓝矮(WBD)、洋葱黄化(OY-W)植原体的TMK-a、TMK-b的氨基酸序列进行了比对和相似性分析,结果显示Pa WBPS TMK-b与WBD TMK-2和OY-W TMK-b之间的相似性分别为95.65%和99.03%;Pa WBPS TMK-a-1与Pa WBPS TMK-a-2的相似性为90.57%,且二者与WBD TMK-1和OY-W TMK-a之间的相似性为87.32%-94.26%;而Pa WBPS、WBD、OY-W三种植原体的TMK-a与TMK-b之间的相似性仅为22.22%-25.95%。构建了Pa WBPS TMK-b、TMK-a-1、TMK-a-2的p ET28a原核表达载体,对Pa WBPS TMK-b、TMK-a-1、TMK-a-2 3种蛋白进行了原核表达,经Ni-NTA柱纯化后,利用双酶法进行了胸苷酸激酶催化活性测定,结果表明,Pa WBPS TMK-b具有较高的胸苷酸激酶活性,为85.96±0.74 U·mg-1,而Pa WBPS TMK-a-1和TMK-a-2几乎没有胸苷酸激酶活性。本文为进一步研究胸苷酸激酶在植原体繁殖过程中的作用机理奠定了基础。     

泡桐丛枝植原体河北平山和江西吉安株系胸苷酸激酶基因多态性分析

设计引物并 PCR扩增河北平山株系 PaWBPs 和江西吉安株系 PaWBJan 的 tmk 基因,直接和克隆测定tmk序列,利用DNAMAN,MEGA等软件进行序列多样性、序列变异、蛋白功能域、系统进化等比较分析。结果表明：从 PaWBPs和 PaWBJan中克隆测序的93条和41条 tmk-a中,分别有52和24种不同的序列,tmk-a ORF 可被划分为2类,tmk-a-1为639 bp,tmk-a-2为627 bp,PaWBPs株系tmk-a-1与tmk-a-2序列条数的比值约为2.5,而PaWBJan株系为3.3; PaWBPs有5个相同的 tmk-a-1序列与 PaWBJan的一个 tmk-a-1序列及枣疯植原体 tmk-Y 序列完全一致。因多位点突变,PaWBPs含假基因比例达48.1%,PaWBJan的为41.7%; tmk-b基因为单一序列,两株系的 tmk-b核酸序列相似性为99.8%,编码蛋白仅有一个氨基酸差异。TMK-a和 TMK-b中都具有胸苷酸激酶的3个保守功能域。系统进化分析显示,泡桐丛枝植原体的2个株系的所有tmk-a序列都与枣疯植原体tmk-X和tmk-Y等聚为一个进化枝Ⅰ中;而tmk-b与小麦蓝矮tmk-2等聚为另一进化枝Ⅲ,基于tmk-b序列和基于16S rDNA序列构建的系统进化树一致,tmk-b有助于植原体16Sr组水平的分类,而 tmk-a可用于植原体株系变异和遗传多样性分析。