长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

Construction of a plasmid containing functional Escherichia coli uvrA, B, and C genes in a configuration potentially suitable for mammalian expression

A plasmid, pUVABC-2, was constructed that encodes functional uvrA, B, and C genes of Escherichia coli. This plasmid also contains the gpt and ampr genes for positive selection in either bacterial or mammalian systems. Each of the uvrA, B, C, and gpt genes is located between SV40 initiation and termination signals and retains the original bacterial promoters. This recombinant vector conferred a wild-type UV resistance phenotype to uvrA-, B-, and C- strains of E. coli. The results indicate that each of the uvr genes contained in pUVABC-2 function in E. coli. The plasmid is a potential biological probe for DNA repair in mammalian cells.     

大鼠肝生长发育过程中ACP、AKP、HSC70/HSP68和PCNA的变化(英文)

磷酸酶（ＡＣＰ、ＡＫＰ）在生物的机能分化中起重要作用,热休克蛋白（ＨＳＰｓ）是近几年发现的一类在胚胎发育、细胞生存中起重要作用的分子,无论是胚胎发育还是细胞结构和功能构建都和细胞增殖密切相关,增殖细胞核抗原（ＰＣＮＡ）是检测细胞增殖的良好指标。 本实验用组织化学、免疫组织化学、Ｗｅｓｔｅｒｎ印迹、酶的原位复性电泳、体视学分析等方法定性和定量分析了酸性磷酸酶（ＡＣＰ）（Ｆｉｇ．１＆２）、碱性磷酸酶（ＡＫＰ）（Ｆｉｇ．４＆５）、构成性热休克蛋白 ７０／诱导性热休克蛋白 ６８（ＨＳＣ７０／ＨＳＰ６８）（Ｆｉｇ．６）和ＰＣＮＡ（Ｆｉｇ．７＆８, Ｔａｂｌｅ１）在大鼠肝生长发育（从１４天胚胎到成体）过程中的动态变化。结果表明：（１）在大鼠肝生长发育过程中,ＡＣＰ有两个活性高峰期,其时段处于大鼠吃奶和吃饲料起始期（Ｆｉｇ．１＆２）;（２）在ＡＣＰ的第一个活性高峰期时,ＡＫＰ活性降低;而在ＡＣＰ的第二个活性高峰期时,正值ＡＫＰ的活性高峰期（Ｆｉｇ．３）;（３）ＡＣＰ活性高峰期也是ＰＣＮＡ含量高峰期;（４）ＨＳＣ７０／ＨＳＰ６８在刚断奶的幼鼠肝和成体肝中表达量较多,其他时段表达极少。根据上述结果推测：ＡＣＰ和ＰＣＮＡ通过调节细     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。     

Identification, mRNA expression and characterization of a novel ANK-like gene from Chinese mitten crab Eriocheir japonica sinensis

Ankyrins are a family of adapter molecules mediating linkages between integral membrane and cytoskeletal proteins. Ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. A novel ANK-like gene of Chinese mitten crab Eriocheir japonica sinensis (denoted as EjsANK) was identified and cloned by expressed sequence tag and rapid amplification of cDNA end approaches. The full-length cDNA of EjsANK is 4375 bp and contains an open reading frame of 1095 bp which encodes a 364 amino acids polypeptide (40.23 kD) bearing seven ankyrin repeats. EjsANK cDNA has a 3073 bp uniquely long 3′ untranslated region with three K-box elements, one GY-like box domain and one Brd-like box domain. Sequence alignment and three-dimensional structural analyses revealed that EjsANK should be a novel cytosolic member of the ankyrin family. Fluorescent real-time quantitative RT-PCR approach was performed to examine the expression profiles of EjsANK mRNA by testing its relative level in three types of tissues at three different developmental stages, respectively. We found that the relative level of EjsANK mRNA expression was significantly higher in the abdomen at the first crab stage. Functional bioinformatics prediction analyses indicated that EjsANK has an analogical effect like IκB which is a key component of IκB/NF-κB complex in mammalian cells playing very important roles in the development process. Results suggest that EjsANK gene is involved in the early developmental regulation of Chinese mitten crab, especially brachyurization regulation.     

沙冬青AmFAD7和AmFAD8基因的克隆与转化及其功能初步分析

对强抗逆植物蒙古沙冬青2个脂肪酸去饱和酶基因AmFAD7和AmFAD8的功能进行了初步研究。结果表明:(1)AmFAD7和AmFAD8的编码蛋白分别含455和457个氨基酸残基。(2)半定量RT-PCR分析显示,在野外生长植株的嫩叶中,AmFAD7表达量在夏季很低、冬季较高,春秋两季略低于冬季,而AmFAD8在春秋两季和初冬时表达量高于其他月份;在低温(4℃~-6℃梯度降温)处理2~48h的幼苗中,AmFAD7的表达呈先降低后升高的变化趋势,而AmFAD8的表达比处理前略有上调;在高温(42℃)处理2~48h的幼苗中,2个基因的表达均被抑制,尤其对AmFAD7的抑制较为迅速而且明显;在干燥处理2~48h的幼苗中,AmFAD7的表达量有较明显的升高,而AmFAD8则略有降低。(3)成功构建了2个基因的植物表达载体(p3300-35S-AmFAD7和p3300-35S-AmFAD8)并转化拟南芥,分别获得18和16个转基因株系。RT-PCR检测表明,其中F7-1和F7-2以及F8-1~F8-4转基因株系中目的基因的表达量均较高。(4)相对电导率分析显示,在正常温度下,AmFAD7转基因株系(F7-1和F7-2)的相对电导率与野生型无显著差异;在低温(-1℃)胁迫24h后,F7-1和F7-2的相对电导率(分别为32.8%和36.1%)显著低于野生型(48.5%),而在高温(37℃)胁迫24h后,F7-1和F7-2的相对电导率(分别为44.7%和41.9%)显著高于野生型(33.2%)。研究表明,AmFAD7在转录水平受低温和干燥胁迫的诱导,但被高温胁迫抑制,而AmFAD8的转录受低温诱导但被干燥和高温胁迫抑制;在拟南芥中组成型表达AmFAD7增强了转基因植株在低温胁迫下细胞膜的稳定性及其耐冻性,但却增加了其在高温胁迫下的细胞膜损伤程度和热敏感性。     

Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Hydration of Oleic Acid by Enterococcus gallinarum, Pediococcus acidilactici andLactobacillus sp. Isolated from the Rumen

Three ruminal bacteria found to convert oleic acid to 10-hydroxystearic acid were identified asEnterococcus gallinarum , Pediococcus acidilactici and a lactobacillus physiologically similar toLactobacillus reuteri and L. fermentum. The oleic acid-hydrating properties of the three bacteria were determined and hydration was shown to be predominantly a feature of anaerobic culture with relatively little hydration occurring in aerobic culture. Hydration was highly pH dependent and not related to cell growth. At the optimal hydration pH for each bacterium, hydration yields were 97%, 93% and 76% for the E. gallinarum, P. acidilactici andLactobacillus strains, respectively. The enterococcus and the lactobacillus hydrated oleic acid only after growth ceased whereas the pediococcus hydrated oleic acid during the late logarithmic growth. Hydration was not specific for oleic acid with all three bacteria hydrating the oleic acid homologue palmitoleic acid. None of the bacteria hydrogenated oleic acid to stearic acid. Our results suggest that a capacity to hydrate oleic acid may be a property of many lactic acid bacteria.     

盐胁迫对大麦叶片类囊体膜组成和功能的影响

盐胁迫下大麦叶片类囊体膜蛋白和叶绿素含量以及对绿素a／叶绿素b、磷脂／膜蛋白和膜脂结合半乳糖／膜蛋白的比值下降,膜脂中亚麻酸（18：3）摩尔百分数上升,不饱和指数上升,类囊体膜H －ATPase活性先升后降,希尔反应一直呈较高活性。类囊体膜组分和功能的变化可能与植物盐适应有关。     

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 – 16 kDa (group 1), 27 – 28 kDa (group 3: chitinases) and 36 – 40 kDa (group 2a: -1,3-glucanases).     

枯草芽孢杆菌L-脯氨酸合成途径中glnA、proB、proA基因功能探究

【目的】从基因水平探究枯草芽孢杆菌渗透压调节因子L-脯氨酸合成途径中glnA、proB、proA基因的功能,通过分子改造实现对代谢途径的人工扰动。【方法】从枯草芽孢杆菌WB600出发,通过向胞内引入一系列基因敲除或过表达,分别构建了proB和proA基因过表达的重组菌WB601和WB602、glnA基因缺失的重组菌WB603以及在此基础之上过表达proB基因的重组菌WB604。借助菌株胞外和胞内游离脯氨酸积累的表型分析影响途径的关键节点。【结果】在非胁迫条件下,重组菌WB601和WB602胞外脯氨酸含量分别是原始菌的2.21倍和2.82倍,单位细胞胞外脯氨酸得率分别是原始菌的4.09倍和9.80倍,胞内游离脯氨酸含量分别是原始菌的1.91倍和3.34倍;重组菌WB603胞外脯氨酸含量上升至1221.43 mg/L,是原始菌的6.28倍,单位细胞胞外和胞内游离脯氨酸得率分别为原始菌的9.13倍和3.66倍;而重组菌WB604胞外脯氨酸含量最高达1391.65 mg/L,相比菌株WB603,其胞外脯氨酸含量及单位细胞得率分别提高了13.94%和14.10%,且胞内游离脯氨酸含量提高了32.60%。在5%Na Cl胁迫条件下,重组菌WB601和WB602的胞外脯氨酸含量分别是原始菌的1.94倍和1.54倍,单位细胞胞外脯氨酸得率分别是原始菌的2.15倍和2.19倍;重组菌WB603胞外脯氨酸含量及其单位细胞得率分别是原始菌的4.16倍和7.29倍;相同条件下,相比于重组菌WB603,重组菌WB604的胞外脯氨酸含量及其单位细胞得率分别提高了32.61%和5.54%。此外,实验组菌株的胞内游离脯氨酸含量均高于非胁迫时,并达到相对平衡状态。【结论】proB和proA基因的过表达均能显著提升细胞合成脯氨酸的能力,并且能增强细胞的耐盐性;glnA基因的缺失能增强脯氨酸合成途径,提高脯氨酸的积累;两种效应的正向叠加可进一步提升细胞脯氨酸合成能力。     

油茶微量元素铜铁锌吸收和积累特征

为弄清油茶(Camellia oleifera)对微量元素铜、铁、锌的吸收利用特征,对5年生‘长林4号’各器官的铜、铁、锌元素含量及其年变化进行了研究。结果表明,油茶植株中铁元素的含量最高,其次为锌和铜元素;单株油茶对锌、铜元素的年积累量分别为62.97 mg和22.60 mg,约为3∶1。从果实发育期至成熟期,锌元素的单株吸收积累量为40.18 mg,约占年吸收积累量的63.81%,从抽梢期至果实成熟期,铜元素的单株吸收积累量为20.04 mg,占年吸收积累量的88.67%,从休眠期至抽梢期,油茶地上部分生长所需的铜、锌元素分别有30.25%和57.90%来源于根系贮存的营养;油茶对铁元素的吸收积累则集中在抽梢期至果实发育期,单株吸收量达0.34 g。这些为指导油茶科学施肥提供了理论依据。     

白番红花CrCBF1基因的克隆及功能分析

该研究选取新疆地区耐寒植物白番红花为研究材料,采用RT-PCR方法,从白番红花中克隆得到白番红花CBF1的全长cDNA序列,命名为CrCBF1(GenBank登录号MF681787)。结果表明,CrCBF1基因完整开放阅读框ORF长642bp,编码213个氨基酸,分子量为23.8kD,理论等电点为4.99,具有CBF家族典型的AP2保守结构域;亚细胞定位分析显示,CrCBF1基因编码的蛋白定位于细胞核;酵母自激活分析显示,CrCBF1转录因子具有转录激活活性;酵母功能验证分析显示,过表达CrCBF1基因可以明显提高酵母的抗寒性。