Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ～30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.     

激动乙醛脱氢酶2对抗糖尿病大鼠心肌缺血/再灌注损伤的作用

目的:探讨激动乙醛脱氢酶2(ALDH2)在糖尿病大鼠心肌损伤中的作用。方法:腹腔注射55 mg/kg链脲佐菌素复制糖尿病大鼠模型,分为糖尿病组和乙醇+糖尿病组(n=8)。8周后行离体心肌缺血/再灌注(I/R),测定心室动力学指标和复灌期间冠脉流出液中乳酸脱氢酶(LDH)含量。测定空腹血糖、糖化血红蛋白(HbA1c)水平。RT-PCR和Western blot测定左心室前壁心尖组织线粒体ALDH2 mRNA和蛋白表达。结果:与正常大鼠心肌I/R相比,糖尿病大鼠左室发展压、左心室最大上升和下降速率、左室做功进一步下降,左室舒张末压抬高,复灌期冠脉流出液中LDH释放增多,心室ALDH2 mRNA和蛋白表达降低;与糖尿病大鼠心肌I/R相比,ALDH2激动剂乙醇明显促进左室发展压、左心室最大上升和下降速率、左室做功的恢复,降低左室舒张末压,同时降低HbA1c水平和LDH的释放,ALDH2 mRNA和蛋白表达增高。结论:糖尿病大鼠心肌缺血/再灌注时,心肌ALDH2表达降低;增强ALDH2在糖尿病大鼠心肌中的表达可发挥保护作用。     

鞘氨醇-1-磷酸对人脐带间充质干细胞增殖及表面标记物表达的影响

鞘氨醇－1－磷酸（sphingosine－1 phosphate, S1P）是一种脂质信号分子,与细胞增殖、凋亡和迁移等有密切关系．本研究发现,S1P促进人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC－MSCs)增殖,但目前关于其作用信号通路及S1P对hUC－MSCs表面标记表达的影响尚不十分清楚．Real－time PCR检测hUC－MSCs中S1P受体mRNA表达情况,发现在hUC－MSCs中优势表达S1PR1 3,而S1PR4、S1PR5的表达很少．MTT法检测S1PR1/3拮抗剂VPC23019、S1PR2拮抗剂JTE013、S1PR3拮抗剂CAY10444、Gi蛋白抑制剂PTX和ERK抑制剂PD98059对S1P诱导hUC－MSCs增殖的影响．结果显示,VPC23019完全抑制S1P诱导的hUC MSCs增殖,JTE013对此没有明显影响,CAY10444部分抑制S1P诱导的hUC MSCs增殖,PTX、PD98059完全抑制S1P诱导hUC－MSCs增殖．进一步用Western印迹检测ERK1/2磷酸化水平揭示,S1P通过促进ERK1/2磷酸化进而促进hUC MSCs增殖．流式细胞术检测发现,S1P对hUC MSCs表面标记物(CD45、CD34、CD90、CD29、CD105、CD44、CD73、CD71)表达没有明显影响．本研究证明,S1P通过S1PR1/3、Gi偶联蛋白及ERK1/2信号通路促进hUC MSCs增殖,而对hUC MSCs表面标记物表达无明显影响．     

CD44+CD24-Λow乳腺癌干细胞的流式分选及其肿瘤干细胞特性的初步鉴定

目的:通过表面标志分选法富集乳腺癌干细胞,并初步鉴定其肿瘤干细胞特性.方法:采用流式细胞分选术从人乳腺癌细胞系MCF-7中分选CD44+CD24-Λow乳腺癌干细胞,并进行干细胞比例分析;用免疫荧光法检测、比较分选获得的细胞和对照细胞的干性和分化标记物Oct-4、SOX-2、CK-18和α-SMA的表达状态.结果:分选获得的CD44+CD24-Λow乳腺癌干细胞阳性比例达90％以上;免疫荧光检测结果显示,CD44+CD24-Λow细胞亚群比non-CD44+CD24-Λow细胞亚群高表达干细胞转录因子Oct-4、SOX-2,低表达分化因子CK-18、α-SMA;体外实验表明,CD44+CD24-Λow细胞亚群具有更强的成球生长能力,并具有双向分化潜能.结论:CD44+CD24-Λow表面标记物分选的方法可以富集高纯度的乳腺癌干细胞,且呈现干性因子Oct-4和SOX-2高表达.     

三疣梭子蟹精卵相互作用的扫描电镜观察

应用扫描电镜技术观察了三疣梭子蟹的精卵相互作用。未受精成熟卵表面较光滑、无受精孔,但有许多微孔。成熟卵外被卵膜,内为卵母细胞。在卵自然产出后,精子迅速发生顶体反应使顶体囊外翻并压入卵膜,而核仍留于卵膜外,核辐射臂不收缩且仍附着于卵膜上。三疣梭子蟹为多精着卵和多精入卵膜。精子外翻顶体囊压入卵膜后,核辐射臂陆续回缩直至消失。作用于顶体丝上的卵母细胞主动拖精作用对入卵膜精子的进一步入卵、受精至关重要,环状卵膜突起的向心伸展也有一定的协助作用。探讨了着卵精子的顶体反应、精子入卵膜的机制及卵子在精子入卵过程中的作用     

原生质体诱变选育乳糖酶高产菌株

采用紫外线诱变和^60Co-γ射线协同诱变的方法,对出发菌株Uco-3的原生质体进行诱变处理,通过正突变率与诱变剂量的相互关系,确定最佳诱变剂量。采用4min的紫外线照射和剂量为500Gy的γ射线对黑曲霉Uco-3的原生质体进行诱变,软得一株产高温乳糖酶的高产突变株,突变株产乳糖酶能力显著提高,产酶活力达44．37U／mL,是出发菌株Uco-3的2．73倍。     

Structural mechanisms underlying nucleotide-dependent self-assembly of tubulin and its relatives

The alphabeta-tubulin dimer assembles into microtubules, essential polymers in all eukaryotic cells. Microtubules are highly dynamic, a property that derives from tubulin's GTPase activity. Both the bacterial homolog, FtsZ, and the recently discovered bacterial tubulins from Prosthecobacter self-assemble in a nucleotide-dependent manner into protofilaments similar to those that form the microtubule wall. A number of structural studies of alphabeta-tubulin, gamma-tubulin (the isoform involved in microtubule nucleation), FtsZ and bacterial tubulin, in a variety of nucleotide and polymerization states, have been reported in the past few years. These studies have revealed the similarities and differences between these structures and their possible functional implications. In particular, a two-state mechanism has been proposed for the recycling of alphabeta-tubulin during the microtubule disassembly-assembly cycle; this mechanism may be unique to eukaryotic dimeric tubulin and the microtubule structure.     

Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway

Early growth response factor-1 (Egr-1) and osteopontin (OPN) play important roles in the migration and proliferation of vascular smooth muscle cells (VSMC), but little is known about their relationship. Therefore, we transfected VSMCs with either Egr-1 cDNA, Opn cDNA, a DNA enzyme designed to target Egr-1 (ED5), or antisense Opn oligodeoxynucleotides and examined changes in Egr-1 and OPN expression at the mRNA and protein levels. OPN expression levels were increased in cells that were stably transfected with Egr-1 cDNA. By contrast, both Egr-1 and OPN expression were reduced when ED5 was transfected into Egr-1-expressing cells. Similarly, Opn transfection upregulated Egr-1 levels, while Opn anti-sense oligodeoxynucleotide transfection decreased Egr-1 expression. ChIP analysis showed that Egr-1 binds to the Opn gene promoter. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor PD98059 inhibited the upregulation of Egr-1 by OPN. We find that Egr-1 and OPN positively regulate each other in VSMCs.     

Characterization and expression profiles of miRNAs in rice seeds

Small RNAs (sRNAs) are common and effective modulators of gene expression in eukaryotic organisms. To characterize the sRNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 797 399 22-nt sequence signatures, of which 111 161 are distinct ones. Analysis on the distributions of sRNAs on chromosomes showed that most sRNAs originate from interspersed repeats that mainly consist of transposable elements, suggesting the major function of sRNAs in rice seeds is transposon silencing. Through integrative analysis, 26 novel miRNAs and 12 miRNA candidates were identified. Further analysis on the expression profiles of the known and novel miRNAs through hybridizing the generated chips revealed that most miRNAs were expressed preferentially in one or two rice tissues. Detailed comparison of the expression patterns of miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated. In addition, differential accumulations of miRNAs and corresponding miRNA*s suggest the functions of miRNA*s other than being passenger strands of mature miRNAs, and in regulating the miRNA functions.