牙龈卟啉单胞菌Hgp44基因的克隆表达与纯化

目的 克隆表达牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)牙龈素中黏附素区域的Hgp44基因,并纯化目的蛋白.方法 通过聚合酶链反应(PCR)和基因重组技术,克隆得到PgATCC33277的Hgp4基因,插入到克隆载体pMD18-T中并测序鉴定.经过酶切后将目的基因片段与表达载体pET22b相连,构建出表达质粒pET22b-Hgp44.将重组质粒转化到感受态细胞BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达融合蛋白,通过SDS聚丙烯酰胺凝胶电泳及蛋白质印迹法进行分析.用固定化金属亲和层析法对重组蛋白进行分离纯化.结果 目的基因片段约为1100 bp,与预期大小相符,测序结果与GenBank中ATCC33277国际标准菌株的RgpA的等位基因序列U15282一致.IPTG诱导后的菌体经SDS聚丙烯酰胺凝胶电泳后形成一个以包涵体形式存在的44 000的融合蛋白.蛋白质印迹法检测证实其具有免疫原性.用镍离子金属螯合层析柱纯化出了目的蛋白,最后得到约3.5 mg/L的目的蛋白.结论 成功克隆表达了PgHgp44基因,并纯化出了目的蛋白.     

Immune responses to trichloroethylene and skin gene expression profiles in Sprague Dawley rats

Objective To characterize the immune reaction in SD rats exposed to trichloroethylene （TCE） and to identify the gene expression profiles involved in skin after TCE exposure. Methods Fifteen percent of TCE was injected intradermally into the rat back （100 μL/120 g） at intervals of 7 days. Whole blood was collected 24 h after the fifth or seventh intradermic administration of TCE. The percentages of CD4＋ and CD8＋ of T lymphocytes were measured by a flow cytometer. The concentrations of IFN-gamma and 1L-4 in the serum were semi-quantified by ELISA. Total RNAs of skin samples at 3 h or 24 h after the seventh dose of TCE in SD rats were extracted, and gene expression proftles of these tissues were analyszed by rat toxicology U34 array of Affymetrix. Results Obvious decline of CD4＋ in T lymphocytes was observed in the TCE-administer group. No significant concentration differences in IFN-gamma and IL-4 were found between TCE-treated and control rats. Gadd45a and Mel were significantly up regulated in skin tissue 24 h after TCE exposure. The expression regulation of immune response factors was as active as proteins associated with lipid metabolism and synthesis process in these skin samples of SD rats exposed to TCE. Conclusion T-helper type 1 cells mediate immune response can not be elicited in TCE-treated SD rats, but certain immune disorder can be induced.     

中度低温体外循环对大鼠海马c-fos,bcl-2及bax mRNA表达的影响

目的:观察中度低温体外循环(CPB)对大鼠海马即刻基因c-fos、凋亡相关基因bcl-2和baxmRNA表达的影响。方法:雄性SD大鼠随机分为CPB组(n=6)及假手术对照组(Sham组,n=6)。所有动物在咪唑安定、芬太尼麻醉后经口插管控制呼吸,置入颈静脉流出管和尾动脉输入管,肝素抗凝(500U/kg)。CPB组采用中度低温CPB(26℃—28℃),经尾动脉灌注、颈静脉右心房-腔静脉引流,灌流量160mL·kg-1·min-1,总转流时间2h,Sham组除不经历CPB外,其余操作同CPB组相同。实验中进行动脉压、ECG及动脉血气监测。术后1h处死动物,立即断头取脑分离海马组织,放入-70℃液氮罐保存。基因mRNA检测采用逆转录多聚酶联反应(RT-PCR)方法。结果:海马c-fos、bcl-2和baxmRNA表达CPB组显著高于Sham组;bax与bcl-2mRNA表达比值CPB组显著高于Sham组。结论:中度低温体外循环可引起海马即刻基因c-fos、凋亡相关基因bcl-2和baxmRNA的表达增加。     

Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies

The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.     

神经细胞黏附分子L1在先天性巨结肠中的表达

目的：研究L1型神经细胞黏附分子（LICAM）基因在先天性巨结肠不同肠段的表达。方法：分别取16例先天性巨结肠惠儿狭窄段和正常段平滑肌组织，经处理后提取总RNA，应用逆转录多聚酶链反应（RT-PCR）扩增目的基因和看家基因片段，观察狭窄段和正常段的LICAM基因的表达，并与看家基因（β-aetin）在狭窄段和正常段的表迭作对比。结果：16例患者正常段LICAM和β-actin均有明显的表达，狭窄段β-actin亦有明显的表达，但LICAM均无表迭或弱表达。结论：先天性巨结肠患者狭窄段LICAM减少的原因可能是LICAM的mRNA的减少或缺如，并进一步引起病变段运动障碍和巨结肠发生。     

The ontogeny of drug metabolism enzymes and implications for adverse drug events

Profound changes in drug metabolizing enzyme (DME) expression occurs during development that impacts the risk of adverse drug events in the fetus and child. A review of our current knowledge suggests individual hepatic DME ontogeny can be categorized into one of three groups. Some enzymes, e.g., CYP3A7, are expressed at their highest level during the first trimester and either remain at high concentrations or decrease during gestation, but are silenced or expressed at low levels within one to two years after birth. SULT1A1 is an example of the second group of DME. These enzymes are expressed at relatively constant levels throughout gestation and minimal changes are observed postnatally. ADH1C is typical of the third DME group that are not expressed or are expressed at low levels in the fetus, usually during the second or third trimester. Substantial increases in enzyme levels are observed within the first one to two years after birth. Combined with our knowledge of other physiological factors during early life stages, knowledge regarding DME ontogeny has permitted the development of robust physiological based pharmacokinetic models and an improved capability to predict drug disposition in pediatric patients. This review will provide an overview of DME developmental expression patterns and discuss some implications of the data with regards to drug therapy. Common themes emerging from our current knowledge also will be discussed. Finally, the review will highlight gaps in knowledge that will be important to advance this field.     

Sustained neural activity to gaze and emotion perception in dynamic social scenes

To understand social interactions, we must decode dynamic social cues from seen faces. Here, we used magnetoencephalography (MEG) to study the neural responses underlying the perception of emotional expressions and gaze direction changes as depicted in an interaction between two agents. Subjects viewed displays of paired faces that first established a social scenario of gazing at each other (mutual attention) or gazing laterally together (deviated group attention) and then dynamically displayed either an angry or happy facial expression. The initial gaze change elicited a significantly larger M170 under the deviated than the mutual attention scenario. At around 400 ms after the dynamic emotion onset, responses at posterior MEG sensors differentiated between emotions, and between 1000 and 2200 ms, left posterior sensors were additionally modulated by social scenario. Moreover, activity on right anterior sensors showed both an early and prolonged interaction between emotion and social scenario. These results suggest that activity in right anterior sensors reflects an early integration of emotion and social attention, while posterior activity first differentiated between emotions only, supporting the view of a dual route for emotion processing. Altogether, our data demonstrate that both transient and sustained neurophysiological responses underlie social processing when observing interactions between others.