Cytoskeleton‐associated proteins are enriched in human embryonic‐stem cell‐derived neuroectodermal spheres

The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2‐DE using protein extracts from hESC‐derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis‐related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton‐associated proteins (CSAP) such as Fascin‐1, Cofilin‐1, and Stathmin‐1. Western‐blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin‐1 and Stathmin‐1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin‐1 in (sub‐)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton.     

定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制

[目的]细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。[方法]首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc² 155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc² 155的胞内药物浓度。[结果]光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc² 155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。[结论]蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。     

SULT1A1 Arg213His polymorphism, smoked meat, and breast cancer risk: a case-control study and meta-analysis

SULT1A1 is involved in both detoxification of estrogens and bioactivation of carcinogens in smoked meat. SULT1A1 Arg213His polymorphism's effect on breast cancer risk is still unclear. We recruited 400 case-control pairs to investigate the association between SULT1A1 genotypes and breast cancer risk, and the combined effect of SULT1A1 polymorphism and daily intake of smoked meat. Participants were questioned about their dietary habits and other risk factors, and their SULT1A1 genotypes were determined. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were estimated by multivariable unconditional logistic regression. We also performed a meta-analysis of relevant published studies to test these associations. In the case-control study, no significant associations were observed between SULT1A1 polymorphism and breast cancer risk. In the meta-analysis, SULT1A1 His/His genotype slightly increased risk among both overall and postmenopausal women (OR(pooled-overall)=1.12, 95% CI: 1.02-1.24; OR(pooled-post)=1.17, 95% CI: 1.03-1.32). A larger positive association was observed in Asian populations (OR(pooled-Asian)=2.01, 95% CI: 1.24-3.26). In our case-control study, high energy-adjusted daily intake of smoked meat was significantly associated with breast cancer risk in overall, pre- and postmenopausal women (aORs: 2.31-3.13, OR 95% CIs exclude 1). High smoked meat intake interacted positively with the His variant allele (all γ>1). These results correlated with those of the meta-analysis (γ(pooled-overall)=1.27). The SULT1A1 His/His genotype may increase the risk of breast cancer among Asian women, and dietary exposure to heterocyclic amines and polycyclic aromatic hydrocarbons, along with the SULT1A1 His/His variant genotype, may synergistically increase the risk of breast cancer.     

The host defense peptide LL‐37 activates the tumor‐suppressing bone morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells

The human cathelicidin LL‐37, a pleiotropic host defense peptide, is down‐regulated in gastric adenocarcinomas. We therefore investigated whether this peptide suppresses gastric cancer growth. LL‐37 lowered gastric cancer cell proliferation and delayed G₁‐S transition in vitro and inhibits the growth of gastric cancer xenograft in vivo. In this connection, LL‐37 increased the tumor‐suppressing bone morphogenetic protein (BMP) signaling, manifested as an increase in BMP4 expression and the subsequent Smad1/5 phosphorylation and the induction of p21^Waf1/Cip1. The anti‐mitogenic effect, Smad1/5 phosphorylation, and p21^Waf1/Cip1 up‐regulation induced by LL‐37 were reversed by the knockdown of BMP receptor II. The activation of BMP signaling was paralleled by the inhibition of chymotrypsin‐like and caspase‐like activity of proteasome. In this regard, proteasome inhibitor MG‐132 mimicked the effect of LL‐37 by up‐regulating BMP4 expression and Smad1/5 phosphorylation. Further analysis of clinical samples revealed that LL‐37 and p21^Waf1/Cip1 mRNA expressions were both down‐regulated in gastric cancer tissues and their expressions were positively correlated. Collectively, we describe for the first time that LL‐37 inhibits gastric cancer cell proliferation through activation of BMP signaling via a proteasome‐dependent mechanism. This unique biological activity may open up novel therapeutic avenue for the treatment of gastric cancer. J. Cell. Physiol. 223: 178–186, 2010. © 2010 Wiley‐Liss, Inc.     

Copper (II) promotes the formation of soluble neurotoxic PrP oligomers in acidic environment

Prion diseases are classically considered to be “amyloid diseases” caused by the deposition of amyloid fibrils in the brain. Recent studies identified soluble oligomers of PrP (prion protein) in damaged neuronal tissue. However, the details of PrP oligomerization are still unclear. In this study, we demonstrate that Cu²⁺ plays a vital role in the formation of soluble PrP oligomers. A Cu²⁺‐triggered structural conversion of PrP (90–231) to a β‐sheet isoform in pH 5.0 buffer was revealed by circular dichroism spectra and fluorescence measurement. Soluble oligomers were isolated by size exclusion chromatography from experimental solutions, allowing atomic force microscopy to reveal their morphology. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and flow cytometry assays demonstrated that oligomeric PrP induced significant damage in and apoptosis of neuroblastoma SK‐N‐SH cells. These results indicate that in an acidic environment, Cu²⁺ promotes the formation of neurotoxic soluble PrP oligomers. J. Cell. Biochem. 111: 627–633, 2010. © 2010 Wiley‐Liss, Inc.     

Global gene expression profile of Orientia tsutsugamushi

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria‐infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.     

肩难产临床分析

目的分析肩难产发生原因,对母婴的影响,处理及预防。方法对我院1988年1月～2006年12月住院分娩产妇中出现的15例肩难产病例进行回顾性总结分析。结果肩难产虽然发生率低,但一旦发生即会造成较严重的分娩并发症,并对母婴产生严重危害,甚至新生儿死亡。结论巨大儿常发生肩难产。肩难产处理不当会发生严重的母婴并发症。产科医师除熟练掌握处理肩难产的各种方法外,更要做产前防范与助产的技巧把握。     

Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo

Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins. In the present report, we show that Nef can interact with calmodulin, the major intracellular receptor for calcium. Coimmunoprecipitation analyses with lysates from the NIH3T3 cell line constitutively expressing the native HIV-1 Nef protein revealed the presence of a stable Nef-calmodulin complex. When lysates from NIH3T3 cells were incubated with calmodulin-agarose beads in the presence of CaCl(2) or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca(2+) signaling. Glutathione S-transferase-Nef fusion protein bound directly to calmodulin with high affinity. Using synthetic peptides based on the N-terminal sequence of Nef, we determined that within a 20-amino-acid N-terminal basic domain was sufficient for calmodulin binding. Furthermore, the myristoylated peptide bound to calmodulin with higher affinity than nonmyris-toylated form. Thus, the N-terminal myristoylation domain of Nef plays an important role in interacting with calmodulin. This domain is highly conserved in several HIV-1 Nef variants and resembles the N-terminal domain of NAP-22/CAP23, a myristoylated calmodulin-binder. These results for the interaction between HIV Nef and calmodulin in the cells suggested that the Nef might interfere with intracellular Ca(2+) signaling through calmodulin-mediated interactions in infected cells.