In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution

Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoproteclant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P <.05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P <.05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that (1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and (2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.     

Colocalization of NADPH-diaphorase activity and certain neuropeptides in the esophagus of opossum (Didelphis virginiana)

Nitric oxide and various neuropeptides in the myenteric plexus regulate esophageal motility. We sought colocalization of nitric oxide synthase and neuropeptides in frozen sections of mid-portion of smoothmuscled opossum esophagus using NADPH-diaphorase activity to mark the synthase and immunoreactivity to detect peptides. The peptides, all with demonstrated physiological activity in this organ, were calcitonin generelated peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. The ExtrAvidin Peroxidase immunostain for each peptide was carried up to the final peroxidase reaction with 3-amino-9-ethylcarbazole. The NADPH-diaphorase reaction was applied with short incubation to provide light staining just before the peroxidase reaction was performed. We examined sections for the proportions of singly and dually labeled nerve cells in the myenteric plexus. NADPH-diaphorase activity was highly colocalized with calcitonin gene-related peptide (59%), galanin (54%), and vasoactive intestinal polypeptide (53%). It showed little colocalization with neuropeptide Y (10%) and substance P (8%). The proportions of all nerve cells containing each of the substances were: NADPH-diaphorase-33%, calcitonin gene-related peptide-30%, galanin-55%, neuropeptide Y-16%, substance P-35%, and vasoactive intestinal polypeptide-58%. We conclude that the nerves responsible for peristalsis in the esophagus may act by releasing nitric oxide along with other inhibitory substances, calcitonin gene-related peptide, galanin, and vasoactive intestinal polypeptide, but not excitatory substances, neuropeptide Y and substance P.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Effect of heavy metals on the methanogenic activity of starch-degrading granules

Summary Heavy metals in electroplating effluent inhibited specific methanogenic activity (SMA) of anaerobic starch-degrading granules. The SMA of granules on the degradation of starch were reduced by 50% when each gram of biomass was in contact individually with 105 mg of zinc, 120 mg of nickel, 180 mg of copper, 310 mg of chromium, or >400 mg of cadmium. Granules had higher toxicity-resistance than flocculent sludge, due to their layered structure.     

Studies on yeast host strains for heterologous P450 expression

The Saccharomyces cerevisiae strain AH22 was capable of human P4501A1 expression without detectable background of yeast P450, unlike ATCC44773. Repeated backcrossing to AH22 produced a strain allowing transformation by vectors carrying various common selectable markers. Background yeast xenobiotic metabolism was observed only with growth on complex medium.     

从水稻根部悬浮培养细胞分离原生质体及植株再生

以长香稻(Oryza sativa L．)(糯稻)的幼嫩种子根为材料,在Ms和N6培养基上诱导产生结构松软而分散的愈伤组织,经过AA液体培养基振荡悬浮培养,悬浮培养细胞经酶解后得到了活性较高的原生质体,试验结果表明这是分离原生质体较理想的材料。在附加2,4-D lmg／L(以下单位同)、KT(激动素)0．2、各氨酰胺876、天门冬酰胺266的Ms琼脂糖培养基中,诱导出了大量愈伤组织,在含KT2、NAA(α-萘乙酸)0．2、zT(玉米素)0．2、cH(水解酪蛋白)1000和4％椰子汁的N6培养基中成功地诱导出了由原生质体再生的植株。当代原生质体再生植株能正常开花、结实、产生种子;染色体数均为二倍性(2n＝24),最显著的特点是结实率低,穗粒数减步、生育期延迟。     

陕西省腹菌纲真菌的聚类分析

对产于陕西省的腹菌纲真菌,共４２个ＯＴＵ＇Ｓ和３６个性状,进行了Ｑ模式聚类分析,结果表明：（１）４２个ＯＴＵ＇Ｓ明显的分为六大类群,与Ｄｒｉｎｇ（１９７３）系统的６个目相吻合。（２）栓皮马勃属与灰包科中包被具拟薄壁层的属相似性较小,支持Ｈａｗｋｓｗｏｒｔｈｅｔａｌ（１９８３）将其作为科级处理的观点。（３）作者认为鬼笔属和竹荪属作为两个独立属处理比较合适。     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

黑曲霉S4生淀粉糖化酶纯化组分的性质及其动力学常数

生淀粉糖化酶是可以水解生淀粉的葡萄糖糖化酶(Ec3.2.1.3,α-1.4-糖苷酶),它是近代工业中极为重要的酶类之一。虽然对生淀粉糖化酶可水解淀粉中所有的D-糖苷键已经清楚,由单糖逆合成寡糖的动力学研究也比较深入,但有关它对各种常用生淀粉材料的水解动力学以及各生淀粉糖化酶组分之间的相互关系的研究却很少。本研究通过在不同温度和pH下,生淀粉糖化酶的三个组分对寡糖和各种生淀粉作用的动力学常数,以及在不同pH值下它们对各生淀粉     

大熊猫消化道消化吸收区段游离面的扫描电镜观察

对大熊猫消化道的消化、吸收区段作扫描电镜观察后表明：（１）胃粘膜上皮细胞排列疏松,细胞表面具微绒毛,（２）十二指肠绒毛呈指状,表面凸凹不乎;绒毛表面具丰富的微绒毛,微绒毛表面粗糙,末端膨大。（３）直肠段具丰富的绒毛结构,表面不平滑,具颗粒状物质,但无微绒毛存在：直肠腺丰富。