鸡Ii结合Rab5a和Rab7b分子的分析

本研究旨在探明鸡恒定链（invariant chain，Ii）与内吞体转运蛋白Rab5a和Rab7b结合的结构域和在细胞内共定位的特征。首先，用PCR和基因突变技术将Ii胞浆区与跨膜区[Ii_{（Cyt-Tra）}]、Ii CLIP （class Ⅱ-associated invariant chain peptide）-三聚体区[Ii_{（CLIP-TRIM）}]和Ii突变体[Ii_{（M81-87aa）}、Ii_{（M91-99aa）}和Ii_{（M81-99aa）}]分别插入pET-32a和pEGFP-C1构建相应的原核和真核重组质粒。其次，将构建的含有绿色荧光蛋白的重组质粒与实验室保存的含有红色荧光Rab5a和Rab7b的重组质粒共转染至人胚胎肾细胞系293 T，观察它们的共定位。将构建的原核重组质粒进行表达和纯化，最后用拉下法和免疫印迹检测Ii与Rab5a和Rab7b的结合域。结果表明，成功构建Ii结构域及Ii突变体的重组质粒。Ii_{（Cyt-Tra）}及Ii突变体均能与Rab5a和Rab7b在细胞内共定位，而Ii_{（CLIP-TRIM）}与空载体却不能。Ii的胞浆区和跨膜区是与Rab5a和Rab7b结合的功能结构域，而不是CLIP与三聚体区。综上所述，鸡Ii与Rab5a和Rab7b共定位和结合的区域是其胞浆区和跨膜区，而不是内质网腔区。这些结果提示Rab分子参与了Ii在胞内细胞器的转运机制，为进一步研究Ii及其载体在细胞内的转运机制和功能提供了新的途径。     

Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco''s modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.     

几种因素对培养小鼠精原干细胞的影响

探讨了小鼠胚胎成纤维细胞饲养层及干细胞因子(SCF)、白血病抑制因子(LIF)、碱性成纤维细胞生长因子(bFGF)等对体外培养小鼠精原干细胞的作用.结果显示,在饲养层上,精原干细胞贴壁时间缩至8～12 h,饲养层具有促进其分裂增殖的作用;培养液中加入不同浓度的SCF、LIF及bFGF,可延长精原干细胞在体外的存活时间,其中加入30μg/LSCF后,其体外存活时间与对照组相比差异显著(P<0.05).     

大肠杆菌耐热肠毒素b及其致动物腹泻的机理

介绍了大肠杆菌耐热肠毒素b（STb）的编码基因、理化性质、免疫学特征、分泌特征、毒性相关氨基酸残基、空间结构、膜受体以及STb致动物腹泻的机理。认为STb是一种十分重要的肠毒素，应进一步加强对STb及其致腹泻机理的研究。     

Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Interaction between the sequence of feeding of hay and concentrate,and boiling of barley on feed intake,the activity of hydrolytic enzymes and fermentation in the hindgut of Arabian mares

The interaction between the sequence of feeding of hay and concentrate and the hydrothermal processing of barley in alleviating concentrate effects on intake, and hindgut fermentation in horses was tested. Six Arabian mares (4–10 years of age, 410 ± 35 kg body weight) were used to evaluate the effects of feeding sequence (FS) and type of barley (TB) on intake, and faecal volatile fatty acids (VFA), activities of α‐amylase (AA: EC 3.2.1.1), carboxymethyl cellulase (CMCase: EC 3.2.1.4), microcrystalline cellulase (MCCase: EC 3.2.1.91) and general filter paper degrading activity (FPD). Mares were offered a ration of air‐dried alfalfa and concentrate (70:30 as‐fed) in four subsequent periods of 14 days including 8 days of adaptation and 6 days of sampling. In each period and each meal, mares received concentrate either 30 min after (HC) or 30 min before (CH) alfalfa hay. Barley was either milled or boiled in water. Rectal samples were grabbed directly from rectum once per period. Mares subjected to CH had higher dry matter intakes than mares under HC regime. The acetate:propionate ratio (A:P ratio) in rectal content was higher with CH than HC. The AA activity was higher under CH than under HC. Mares fed boiled barley had lower rectal concentrations of VFA and propionate and a higher A:P ratio than mares fed milled barley. Furthermore, the rectal content showed a higher MCCase activity but a lower AA activity when mares were fed boiled compared with milled barley. Interactions between FS and TB were observed with respect to CMCase activity, and concentrations of propionate and valerate. In conclusion, the present results suggest that both, feeding concentrate before hay and boiling the barley, might improve the hindgut environment in Arabian mares, and that the two measures were mostly additive and sometimes even synergistic.     

江苏省4个太湖新银鱼种群遗传多样性和遗传结构分析

太湖新银鱼(Neosalanx taihuensis)是我国特有的银鱼种类,主要分布在长江和淮河中下游及其附属湖泊,近年来其资源量呈明显下降趋势。为了解太湖新银鱼遗传背景,本研究采用线粒体细胞色素b (Cytochrome b, Cyt b)基因序列,分析了江苏省太湖、高邮湖、洪泽湖和骆马湖4个太湖新银鱼野生群体共144尾样本的遗传多样性及遗传结构。结果显示,太湖新银鱼Cyt b基因序列共发现29个变异位点,定义25个单倍型;平均单倍型多样性(Hd)为0.682±0.037,核苷酸多样性(π)为0.00231±0.00021;4个群体中,高邮湖群体的遗传多样性最高(Hd: 0.609±0.078; π: 0.00094± 0.00027),太湖群体的遗传多样性最低(Hd: 0.343±0.107; π: 0.00075±0.00033)。分子方差分析(AMOVA)显示,太湖新银鱼群体间遗传差异(71.53%)大于群体内遗传差异(28.47%),遗传变异主要来自于群体间。遗传分化指数Fst值统计检验表明,骆马湖群体与太湖、高邮湖和洪泽湖群体之间有显著性差异。分子系统树和单倍型网络进化图分析显示,25个单倍型形成2个明显的地理分支,一支由太湖群体、高邮湖群体和洪泽湖群体组成,另一支由骆马湖群体组成。中性检验和错配分布图分析表明,太湖新银鱼历史上发生过群体扩张。整体来看,太湖新银鱼野生种群遗传多样性较低,应加强种质资源保护。建议将太湖、高邮湖群体和洪泽湖群体作为整体进行管理和保护,骆马湖群体单独管理和保护。