A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.     

Smad泛素化调节因子2siRNA的筛选及转染原代肾小管上皮细胞条件的优化简

试验旨在优化Smad泛素化调节因子2（Smurf2） siRNA最佳转染条件,筛选最佳siRNA干扰片段,进而实现对Smurf2基因的瞬时沉默,为研究Smurf2基因在马兜铃酸肾病（aristolochic acid nephrohathy,AAN）中的作用奠定基础。本研究通过培养小鼠原代肾小管上皮细胞（renal tubular epithelial cells,RTECs）,并以脂质体Lipofectamine^TM 2000为转染介质,将Smurf2 siRNA转染入RTECs;通过观察绿色荧光的表达量及Real-time PCR反应优化转染条件,转染后Real-time PCR检测mRNA表达抑制率。CCK-8法检测Smurf2 siRNA复合物对RTECs活性的影响;同时,用Real-time PCR和Western blotting检测不同位点Smurf2 siRNA对Smurf2 表达的影响。结果显示,当Lipofectamine ^TM 2000与siRNA比例为1.5 μL∶30 pmol时,转染效率最高,为70%～80%;Smurf2-619 siRNA干扰效果最明显;与正常组相比,转染siRNA组的Smurf2蛋白表达水平显著下降（P<0.05）。最终确定了Smurf2 siRNA最佳转染条件,其中Smurf2-619 siRNA对Smurf2 表达的抑制率最高。     

Effect of diacylglycerol acyltransferase 2overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation

Background：Fatty acid（FA） composition is the most important parameter affecting the flavor and nutritional value of the meat.The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2（DGAT2）.The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes,However,little is known about the effect of DGAT2 on the FA composition of these cells.Methods：To investigate the role of DGAT2 in regulating lipid accumulation,FA composition and the expression of adipogenic genes,we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2.Cells were then cultured in differentiation medium（DM） without FA,with a mixture of FAs（FA-DM）,or containing a ~（13）C stable isotope-labeled FA mixture（IFA-DM）.The FA composition of adipocytes was analyzed by gas chromatography-mass spectrometry and gas chromatography-isotope ratio mass spectrometry.Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d.Results：The triacylglyceride（TAG） content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells.When cultured in DM or FA-DM for 12 d,cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs（C16：1 and C18：1）.However,when cells overexpressing DGAT2 were cultured with FA-DM for30 min,the FA composition was almost identical to that of controls.Further,the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d.These results collectively indicate that the higher proportion of mono-unsaturated FAs,C16：1 and C18：1,may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium.This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis（ACACA,FASN,SCD1,and A-FABP?     

Some proteolytic properties of hatching liquid in the sea trout,Salmo trutta m. trutta L.

Proteolytic activity of sea trout hatching liquid was examined towards casein and azocazein as a function of pH and temperature. The optimum pH for caseinolytic and azocaseinolytic activities were 9.4, and 9.0, respectively. At alkaline pH the enzyme was activated by low concentrations of Zn²⁺ ions (10⁻⁵ M). Maximum proteolytic activity of the hatching liquid was observed at 25°C. Temperatures exceeding 30°C caused a rapid reduction in enzyme activity. Proteolytic activity observed at 10°C was approximately 50% of that observed at 25°C. In general, a pseudo-Arrhenius plot indicated a Q₁₀ of 1.6 between 6 and 25°C.     

Presence and ontogeny of intestinal and pancreatic phospholipase A2-like proteins in the Red Sea bream,Pagrus major. An immunocytochemical study

We have studied the location and the ontogeny of the digestive enzyme, phospholipase A₂ (PLA₂) immunohistochemically in the adult and larvae/juvenile of the red sea breamPagrus major by using an antiserum against theNaja naja venom PLA₂. The antiserum reacts with at least one enzyme among the PLA₂s purified from the fish hepatopancreas or intestine. Although the reactivities were comparatively low, it labelled zymogen granules of the pancreatic acinar cells and secretory materials of certain epithelial cells in the depths of epithelial crypts in the pyloric caeca of the adult. The immunoreactivities of PLA₂s were investigated in the viscera of larvae and juveniles of the 0 to 85^th day after hatch. In the larvae of the 13^th day, accumulation of PLA₂-positive zymogen granules in the pancreatic acinar cells were first recognized by the immunostaining. The intensity of the labelling subsequently became stronger and dramatically increased between the 20^th and 30^th day. This increase appeared to be one of the physiological changes associated with the transition to a new benthic life as juveniles. Lack of PLA₂ in the pancreas before the 13^th day may suggest the possibility that larvae utilized exogenous PLA₂, inherent in their prey, to digest the phospholipids. On the other hand, no reactivity was found in the intestine until the 85^th day.     

Oxygen uptake in the Antarctic teleost Pagothenia borchgrevinki. Limitations imposed by X-cell gill disease

Fish in a population of Pagothenia borchgrevinki in McMurdo Sound, Antarctica, are affected by a gill disease (X-cell disease) which causes tissue hyperplasia that results in a decreased gill surface area and an increased water/blood diffusion distance. P. borchgrevinki acquires 95% of its oxygen via the gills, but damage to the gills by X-cell disease did not affect this function. There was no compensatory shift to cutaneous respiration. X-cell disease reduced the ability for oxygen uptake at low ambient P_{O 2} and the decreased uptake was related to the extent of the disease. _{O 2} max was greatly reduced in X-cell affected fish and substantially reduced their aerobic potential. This effect may impair the ability of diseased fish to catch prey and avoid predators.     

牙鲆"鲆优2号"不同养殖地点生长和存活性状的基因型与环境互作分析

为比较牙鲆"鲆优2号"在不同养殖地区的生长和存活性能,实验利用连续多代对生长性状和抗迟缓爱德华氏菌病性状遗传参数评估和基因组选择的结果筛选出的亲本,建立28个"鲆优2号"家系,在河北(Site 1)和山东(Site 2)进行对比养殖试验,利用混合线性动物模型对生长和存活性状进行了基因型与环境互作分析。Site 1和Site 2的平均日增重分别为1.5和1.2 g/d,养殖成活率分别为81.4%和82.2%,"鲆优2号"在两个养殖地点的生长和抗病性能均表现优异。不同养殖环境间收获体质量和存活性状的遗传相关分别为0.57(<0.7)和0.82(>0.7),说明不同养殖环境间收获体质量存在显著的基因型与环境互作效应,但是不同养殖环境间存活性状的基因型与环境互作效应不显著。研究表明,牙鲆"鲆优2号"新品种在不同养殖地点的生长和存活性能均表现良好,为保证良好的推广效果,需要对牙鲆的制种方案进一步优化,针对不同的养殖地区进行"鲆优2号"苗种生产,或培育具有普适性的"鲆优2号"苗种,保证在不同养殖环境下的快速生长和高存活率优势。     

NR/ENR/SiO_2复合材料的阻尼性能研究

采用机械混炼的方法制备不同ENR含量的NR/ENR/SiO2复合材料,使用动态热机械分析仪(DMA)和橡胶加工分析仪(RPA)对NR/ENR/SiO2复合材料的动态力学性能和阻尼性能进行分析。结果表明:ENR对NR/ENR/SiO2复合材料的阻尼性能具有显著影响。动态热机械分析测试结果表明,随着ENR用量的增多,NR/ENR/SiO2复合材料在使用温度范围内的损耗因子积分面积增大,阻尼性能提高。橡胶加工分析频率扫描和应变扫描测试结果表明,在频率小于10 HZ不同应变时,加入了ENR的NR/ENR/SiO2复合材料的阻尼因子均高于NR/SiO2复合材料。扫描电镜分析结果证实,ENR的加入减少了SiO2的自聚,改善了填料在橡胶基体中的分散,从而使NR/ENR/SiO2复合材料力学性能得到提高。     

国家自然科学基金项目（J1210056）

为探讨硫化氢(H2S)作为潜在的新型氮肥增效剂的可能性,通过水培实验,以扬麦16为材料,以NaHS为H2S供体(浓度0.01mmol·L-1),研究了外源低浓度H2S对低氮(2.5 mmol·L-1)、中氮(7.5)和高氮(15 mmol·L-1)条件下小麦幼苗光合作用、硝态氮吸收和同化的影响.结果表明,外源低浓度H2S促进了小麦幼苗的光合作用及对硝态氮的吸收,使低氮条件下植株干物质重增加15.5％,显著提高叶片可溶性蛋白、总氮及叶绿素含量.外源H2S处理后,低氮和中氮水平下叶片硝酸还原酶(NR)、谷氨酰胺合成酶(GS)以及谷氨酸脱氢酶(GDH)活性有所增加,尤其是在低氮水平下增加显著,但在高氮水平下NR与GS活性分别下降22％和13％,GDH活性与对照无明显差异.以上结果说明外源低浓度H2S可提高小麦幼苗对低氮的适应性,促进其生长及对氮素吸收与同化.