仔猪前脑基底部内生长抑素mRNA的分布定位──原位杂交法研究

用原位杂交法研究了５只仔猪前脑基底部一些结构内生长抑素ｍＲＮＡ的分布。结果表明，生长抑素ｍＲＮＡ分布于尾状核、伏隔核、壳核、屏状核、隔核、杏仁核、嗅结节和梨状叶。含生长抑素ｍＲＮＡ的神经元胞体多呈卵圆形或梭形。尾状核和伏隔核内的标记细胞分布较均匀（尾状核前部未观察）。壳核、屏状核、梨状叶和嗅结节内的多位于深层。隔核和杏仁核内出现的标记细胞较少，隔核内的分布于隔外侧核。在猪体上的实验结果与用免疫组织化学法在绵羊和马体上获得的结果有一些差异。     

Changes in carbohydrate levels and their metabolic enzymes in leaves,phloem sap and mesocarp during cucumber (Cucumis sativus L.) fruit development

Levels of carbohydrates and activities of metabolic enzymes were examined in leaves (source), phloem sap (flow) and mesocarp tissues (sink) in the course of cucumber (Cucumis sativus L.) fruit development, from 2 days before anthesis to 20 days after anthesis. While total sugar levels increased in all the three sampling organs, starch levels declined in leaves and mesocarp tissues as fruit development progressed. Glucose and fructose were the primary contributors to the soluble sugar pools in mature leaves. Stachyose was found as the most important component of the phloem sap extracts, followed by sucrose and raffinose. However, the primary sugars accumulated in mesocarp tissues were glucose and fructose, not stachyose or sucrose. Activities of sucrose synthesizing enzymes (sucrose phosphate synthase plus sucrose synthase in the synthesizing direction) exceeded that of sucrose degrading enzymes (acid invertase, neutral invertase plus sucrose synthase in the degrading direction) in leaves, which might cause a sucrose pool utilized in raffinose and stachyose biosynthesis. While alkaline a-galactosidase form I activity declined, stachyose synthase activity showed a rapid increase until 12 days after anthesis and only subsequently decreased in leaves. Activities of sucrose degrading enzymes were always much higher than that of sucrose synthesizing enzymes in mesocarp tissues. Thus, sucrose accumulation could not occur in mesocarp tissues. While stachyose synthase activity steadily decreased, alkaline a-galactosidase form I activity showed a moderate increase before decrease in mesocarp tissues. The relationship between levels of soluble sugars and activities of relative enzymes was also discussed.     

鱼类致病性迟钝爱德华氏菌中多药抗性质粒pEIB202的消除

[目的]考察迟钝爱德华氏菌（Edwardsiella tarda）EIB202中大质粒pEIB202在其致病过程中的作用,将质粒pEIB202消除,为开发抗迟钝爱德华氏菌病的安全减毒的活疫苗打下良好基础。[方法]采用同源重组技术以sacB为反向筛选标记消除质粒。[结果]对质粒pEIB202进行序列分析,发现该质粒编码多种抗性基因及部分VI型分泌系统（T4SS）组分,暗示该质粒可能与E.tarda的多重耐药性及致病力相关;质粒消除菌株EIB202Δp丧失氯霉素及四环素抗性,在生长、毒力、胞外蛋白分泌等方面与野生株无显著差异。[结论]PEIB202质粒是造成EIB202多重耐药性的主要原因,在其致病过程中可能并不起直接作用。     

The Xanthomonas oryzae pv. oryzae type IV pilus alignment subcomplex protein PilN contributes to regulation of bacterial surface-associated behaviours and T3SS system

The gram-negative plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) is able to infect the host rice and effectively colonize in vascular tissues. The type IV pilus (T4P) is one of the major virulence factors playing an important role in migration of Xoo through host vascular tissues. Here, we identified PilN, a T4P alignment subcomplex protein, which is involved in regulation of swimming motility, and analysed its contribution to bacterial surface-associated behaviours and virulence. We found that the pilN deletion mutant exhibited dramatically reduced twitching motility and scarcely detectable levels of T4P major pili PilA, as well as enhanced biofilm formation and exopolysaccharide (EPS) production. In addition, deletion of the pilN gene in Xoo resulted in impaired virulence in host rice and attenuated type III secretion system (T3SS) genes expression, which is independent of PilA assembly. Expression of the relevant pilN gene in trans was capable of restoring twitching motility and biofilm formation to the wild-type levels in the pilN mutant but partially recovering EPS production and virulence. Moreover, the expression of trh and xrvA genes, which encode the HrpG positive regulators, was decreased in the pilN mutant. Our results suggest that PilN executes versatile functions in bacterial virulence and cell surface-associated behaviours.     

GM-CSF与生长抑素融合表达质粒对小鼠肌肉组织GHR和IGF-I mRNA表达的影响

选择70只小白鼠,随机分为7组,每组10只,雌雄各半。其中第1组为对照组,第2-4组分别肌肉注射pcS/2SS、pGM-CSF/SS和pGM-CSF+pcS/2SS DNA疫苗;第5～7组分别口服以减毒沙门氏菌为载体的pcS/2SS、pGM-CSF/SS和pGM-CSF+pcS/2SS DNA疫苗,以β-actin作为内参,利用相对半定量RT-PCR,检测小鼠肌肉组织GHR和IGF-I mRNA的表达。结果表明:第3、5和6组的GHR mRNA表达显著高于1组和7组(P<0.05),第5和6组的IGF-I mRNA表达显著高于1组、2组、4组和7组(P<0.05),GM-CSF与SS的融合表达质粒(pGM-CSF/SS)的GHR和IGF-I mRNA表达高于pGM-CSF+pcS/2SS共同免疫,同种DNA疫苗,口服免疫组的GHR和IGF-I mRNA表达总体上比肌肉注射组要高。这些结果证明,GM-CSF可促进SS DNA疫苗的免疫效果,提高肌肉组织中GHR和IGF-I mRNA的表达;pGM-CSF/SS效果优于pGM-CSF+pcS/2SS,口服免疫组要优于肌肉注射免疫组。     

Tn5 transposon mutagenesis in Acidovorax citrulli for identification of genes required for pathogenicity on cucumber

An Acidovorax citrulli–cucumber pathosystem was established through which A. citrulli mutants with altered pathogenicity, generated by transposon mutagenesis, were identified on cucumber cotyledons. The A. citrulli group I strain FC440 was shown to grow faster in cucumber leaf tissues than a group II strain and was used for Tn5 transposon mutagenesis. A total of 2100 Tn5 insertional mutants were generated, and analysis of the mutant library showed that the transposon insertions were single, independent and stable. A conserved non‐flagellar type III secretion system (NF‐T3SS) ATPase gene hrcN was identified and confirmed to be essential for pathogenicity and functionality of NF‐T3SS in A. citrulli. Comparative sequence analysis of the HrcN protein and its homologues in other representative bacterial plant pathogens revealed that the NF‐T3SS of A. citrulli is close to that of Ralstonia solanacearum and Xanthomonas campestris, but distant from that of Pseudomonas syringae and Erwinia amylovora. The generated Tn5 insertional mutant collection is valuable for identification of genes required for A. citrulli pathogenesis, and the established A. citrulli–cucumber pathosystem will facilitate an improved understanding of A. citrulli biology and pathology.     

粘膜免疫对鸡十二指肠SS mRNA表达的影响

应用鸡新城疫弱毒疫苗进行消化道粘膜免疫,或在肌肉注射生长抑素(SS)亚单位疫苗的基础上应用鸡新城疫弱毒疫苗进行消化道粘膜免疫,通过RT PCR方法检测免疫鸡十二指肠中SS基因的表达.结果表明,首免后第3周,新城疫免疫组的SS mRNA表达高于SS亚单位苗组,但各试验组无显著差异;首免后第4周,新城疫免疫组SS mRNA的表达显著高于SS亚单位苗组(P＜0.05)并高于对照组.提示粘膜免疫可促进小肠粘膜SS mRNA的表达,SS亚单位苗可在基因水平上降低SS mRNA的表达.     

黏膜免疫和半胱胺对十二指肠生长抑素mRNA表达的影响

应用鸡新城疫弱毒苗单一口服和分别灌喂两种剂量半胱胺后再口服鸡新城疫弱毒苗,进行消化道黏膜免疫肉鸡,通过RT-PCR方法检测十二指肠中SS基因表达。结果表明,首免第4周后各个试验组SSmRNA的表达无显著差异。首免第5周后两种剂量半胱胺结合新城疫免疫组SSmRNA的表达比对照组显著增加(P<0.05),单一新城疫免疫组SSmRNA的表达虽然比对照组增加,但并不显著。提示黏膜免疫后淋巴细胞产生的细胞因子可刺激十二指肠SSmRNA的表达,半胱胺可增加十二指肠SSmRNA的表达。肠黏膜免疫可影响动物体内收稿日期:2002-06-17课题来源:国家自然科技基金(30070564)作者简介:杨　倩(1962-),女,南京农业大学动物医学院副教授,博士,主要从事动物免疫神经内分泌研究工作。神经内分泌系统中SSmRNA的表达。     

γ-HCH-Decomposing ability of several strains of Pseudomonas paucimobilis

Abstract

Extract

We succeeded in isolating a γ-HCH (γ-1,2,3,4,5,6-hexachlorocyclohexane)-decomposing aerobic bacterium from an upland field where γ-HCH had been repeatedly applied for more than 10 years. The bacterium was identified as Pseudomonas paucimobilis (Oyaizu and Komagata, 1983; Senoo and Wada 1989; Wada et al. 1989). We assumed that P. paucimobilis was able to thrive in the upland field by acquiring the γ-HCH-decomposing ability (Tu 1975, 1976) based on the following mechanism:     

影响生长抑素重组质粒pEGS/2SS转染HeLa细胞效果的因素研究

旨在分析生长抑素重组质粒pEGS/2SS转染HeLa细胞(人宫颈癌上皮细胞)转染效果的影响因素,探讨最佳转染条件,为进一步研究生长抑素基因疫苗的作用机制和作用效果奠定基础。以脂质体转染法将带有绿色荧光蛋白(GFP)报告基因的重组质粒pEGS/2SS转染HeLa细胞,探讨质粒转染HeLa细胞最佳条件的4个参数:质粒DNA剂量、脂质体剂量、最佳转染时间和质粒提取方法,在荧光显微镜下观察细胞转染情况并计算转染率。结果:在六孔细胞培养板上,当DNA/脂质体剂量为1μg/6μg时,转染效率最高,在转染后72 h达到34.02%;当用Plasmid Maxi Kit试剂盒提取质粒,转染时间为48 h,转染效率最高,达到17.88%。重组质粒pEGS/2SS转染He-La细胞条件是:采用试剂盒(Plasmid Maxi Kit)提取的质粒,DNA和脂质体的剂量分别为1μg与6μg,转染时间48 h,此时的转染效率最高,达17.88%。