The PTB domain of ShcA couples receptor activation to the cytoskeletal regulator IQGAP1

Adaptor proteins respond to stimuli and recruit downstream complexes using interactions conferred by associated protein domains and linear motifs. The ShcA adaptor contains two phosphotyrosine recognition modules responsible for binding activated receptors, resulting in the subsequent recruitment of Grb2 and activation of Ras/MAPK. However, there is evidence that Grb2‐independent signalling from ShcA has an important role in development. Using mass spectrometry, we identified the multidomain scaffold IQGAP1 as a ShcA‐interacting protein. IQGAP1 and ShcA co‐precipitate and are co‐recruited to membrane ruffles induced by activated receptors of the ErbB family, and a reduction in ShcA protein levels inhibits the formation of lamellipodia. We used NMR to characterize a direct, non‐canonical ShcA PTB domain interaction with a helical fragment from the IQGAP1 N‐terminal region that is pTyr‐independent. This interaction is mutually exclusive with binding to a more conventional PTB domain peptide ligand from PTP–PEST. ShcA‐mediated recruitment of IQGAP1 may have an important role in cytoskeletal reorganization downstream of activated receptors at the cell surface.     

MST1 promotes apoptosis through phosphorylation of histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphorylates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phosphorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphorylation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.     

拟南芥转录因子GRAS 家族基因群响应渗透和干旱胁迫的初步探索

GRAS家族是一类植物特有的转录调控因子, 已有报道表明该家族基因在植物生长发育和光信号转导过程中具有重要作用。目前在拟南芥(Arabidopsis thaliana)基因组中已鉴定了33个GRAS家族基因。利用功能基因组学和生物信息学手段,通过基因芯片数据挖掘和基因功能预测, 对拟南芥GRAS家族基因在渗透和干旱胁迫过程中的应答模式进行了初步探索, 提出了一类响应渗透胁迫和干旱胁迫的拟南芥GRAS家族基因。以SCL13为例, 利用基因芯片相关性和GO分析, 对其在渗透胁迫信号转导过程中可能的调控机制进行了预测和分析。这一研究将为阐明GRAS家族基因参与水分胁迫的分子机制提供新的思路, 同时也为植物抗逆分子育种提供候选基因。     

Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats

Background information. AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0–12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly¹⁰³ > Asp¹⁰³) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291 , G1081–G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild‐type) animals. Results. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP‐AQP5s) and expressed in polarized MDCK‐II (Madin—Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP‐AQP5 being translocated less efficiently. Thapsigargin and H‐89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP‐AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co‐localized with LAMP2 (lysosome‐associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co‐localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Conclusion. Replacement of highly conserved hydrophobic Gly¹⁰³ with strongly hydrophilic Asp¹⁰³ in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.     

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.     

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV), a member of the membrane‐containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo‐microscopy (cryo‐EM), we determined the structure of an attenuated vaccine strain, TC‐83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.     

不同肝病变组织中CD34、CD31、Ki-67的表达及意义

目的比较正常肝组织、慢性肝炎、肝硬化、肝细胞肝癌组织及肝转移腺癌中CD34、CD31、Ki-67不同表达,寻找有助于鉴别不同性质病变的生物学标记物.方法正常肝及病变肝组织标本共104例;其中,正常肝组织10例;慢生C型肝炎组织73例;肝硬化组织7例;肝细胞肝癌7例;结肠癌肝转移5例;乳腺癌肝转移2例.73例慢性C型肝炎组织全部为肝穿活检标本,其余组织均为手术切除标本.所有病例标本分别行CD34、CD31、Ki-67免疫组织化学染色,半定量评分系统评价染色结果.统计学分析结果数据.结果在非肿瘤组织,抗CD34阳性染色主要存在于汇管区,亦可见于汇管区周围的肝实质内血窦.阳性染色内皮细胞呈点状、线状、半环状及环状,散在或簇状分布.肿瘤组织内抗CD34阳性染色特征与非肿瘤组织相似,阳性染色血管在肿瘤组织内散布分布.CD34指数在各病变组中的表达排列顺序依次为:肝细胞肝癌>乳腺癌肝转移>结肠癌肝转移>肝硬化>慢性C型肝炎>正常肝组织,从正常肝组织至慢性肝炎至肝细胞肝癌,CD34表达明显增强.组织中,抗CD31阳性染色分布、定位、形态特征与CD34相似.CD31在慢性肝炎、肝硬化、肝细胞肝癌、结肠癌肝转移及乳腺癌肝转移组织中阳性表达率分别为:6.8%(5/73)、100%(7/7)、100%(7/7)、100%(5/5)、100%(2/2);肝癌组织中CD31染色强度明显大于非癌组织中,组间比较具有显著差异(P<0.05).Ki-67阳性染色细胞呈棕黄色核着色,散在分布于肝实质内.阳性染色细胞无形态特殊性,亦无分布上的特殊性.Ki-67在各病变组间的阳性表达率分别为:64.4%(47/73)、28.6%(2/7)、100%(7/7)、100%(5/5)、100%(2/2),其中以在结肠癌肝转移组织中表达最明显;组间比较具有非常显著差异(P<0.05).在正常肝脏、慢性C型肝炎、肝硬化、肝细胞肝癌CD34、CD31、Ki-67三种生物学标记物在同一标本同时表达的阳性率分别为:0%(0/0)、4.1%(3/73)、28.6%(2/7)、100%(7/7),CD34、CD31、Ki-67其中任两种同时表达的阳性率分别为0%(0/10)、63.0%(46/73)、100%(7/7)、100%(7/7).结论 CD34是慢性肝病、肝癌临床病理评价的指标之一,CD34与CD31、Ki-67同时分析有助于建立可靠的诊断.     

左侧视网膜剥夺鸽左前脑差异表达基因全长cDNA的克隆

利用cDNA末端快速扩增 (RACE)技术克隆了一条由左侧视网膜剥夺造成左前脑差异表达EST片段的全长cDNA序列 ,同源性比较后认定 ,其为鸽e(r)基因 .该基因与人类及斑马鱼等脊椎动物e(r)基因的同源性非常高 ,但C端有明显差异 .RNA斑点杂交、逆转录聚合酶链反应 (RT PCR)及Northern印迹等方法检测 ,该基因在不同组织中表达量有差异 .结果表明 ,鸽e(r)基因在左眼视网膜剥夺鸽的左前脑及肝、肾中的表达量较高 ,该基因的表达具有一定的组织表达特异性 .     

Influence of ammonium and nitrate supply on growth,nitrate reductase activity and N-use efficiency in a natural hybrid pine and its parents

Aims Selection of tree species with a high capacity to assimilate N and efficiently utilize N resources would facilitate the success of initial tree seedling establishment in infertile soils. The preference for N forms was tested using three pine species (Pinus densata, Pinus tabuliformis and Pinus yunnanensis). Pinus densata is a natural diploid hybrid between P. tabuliformis and P. yunnanensis .Methods Seedlings of three pine species were supplied with nitrate-N, ammonium-N (at two different pH regimes) or combined ammonium and nitrate as a nitrogen source in perlite culture in a controlled environment.Important findings Seedlings of P. densata had higher total biomass and net photosynthesis when supplied with nitrate-N and ammonium nitrate than with ammonium-N. In parental species, total biomass and net photosynthesis for P. yunnanensis seedlings was higher in ammonium-N than in nitrate-N, whereas the other parental species P. tabuliformis had the highest total biomass among species for all treatments except ammonium with CaCO 3. Most morphological traits in P. densata seedlings were intermediate between its two parental species. However, N-use efficiency and photosynthetic N-use efficiency of P. densata significantly exceeded both parents when supplied with nitrate-N and ammonium nitrate. The results suggested that the diploid hybrid tree species P. densata has a preference for nitrate and is not well adapted to ammonium-N as a sole nitrogen source regardless of the growth medium pH. Based on changes in environmental conditions, such as predicted future temperature increases in high altitude areas associated with climate change, P. densata is likely to be increasingly competitive and have wide adaptation in high altitude regions.