滇水金凤转录因子TT8的克隆及表达分析

为探究滇水金凤(Impatiens uliginosa L.)TT8(TRANSPARENT TESTA 8)基因的功能和表达特性,并解析其对滇水金凤花色的影响,研究以滇水金凤花器官为材料,通过RT-PCR等技术克隆IuTT8基因,并对其进行生物信息学分析;利用qRT-PCR分析该基因在不同花色和不同花发育阶段的表达模式。结果表明,(1)成功克隆得到滇水金凤IuTT8基因,其编码区全长为2 136 bp,编码711 aa,为亲水性不稳定蛋白,gDNA全长为3 938 bp,共有6个内含子;结构域分析发现该蛋白属于bHLH超家族成员,与喜马拉雅凤仙花、山茶等物种的TT8蛋白同源且Motif基序相似。(2)IuTT8与同属植物喜马拉雅凤仙花的聚在一支,相似性约86.34%;多序列比对和系统进化分析显示TT8蛋白的结构域高度保守。(3)IuTT8基因在4种不同花色滇水金凤及其4个不同发育阶段均有表达,除白色外,其表达量均随花发育的进行呈先升后降的趋势;且IuTT8基因的表达量与花色呈正相关,其中以深红色表达量最高,白色表达量最低,深红色S3的表达量约为白色S2时期的48倍。研究表明滇水金凤I...     

Nitric oxide protects cardiac sarcolemmal membrane enzyme function and ion active transport against ischemia-induced inactivation

Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart.     

肇庆星湖湿地可培养放线菌多样性

摘要:【目的】探究星湖湿地可培养放线菌物种多样性,筛选潜在药源活性代谢产物产生菌,为后续菌种资源开发奠定基础。【方法】采用5种选择性分离培养基分离星湖湿地底泥中的放线菌,通过16S rRNA基因同源性分析代表性菌株的物种多样性;以3株病原细菌为指示菌检测分离菌株的抑菌活性;PCR扩增代表菌株的聚酮合酶(PKS I、PKS II)基因、非核糖体多肽合成酶(NRPS)基因、安莎类化合物(AHBA)基因及3-羟基-3-甲基戊二酰辅酶A还原酶(HMGA)基因。【结果】分离到135株放线菌菌株,被鉴定为放线菌纲的7 个目、10个科、13个属,优势类群为链霉菌、小单孢菌及诺卡氏菌。83株检测菌中,24.09%抗金黄色葡萄球菌(Staphylococcus aureus),4.8%抗大肠杆菌(Escherichia coli);24株高活性菌株中PKS I阳性率16.7%,PKS II阳性率62.5%,NRPS阳性率16.7%,AHBA阳性率12.5%,HMGA阳性率29.2%。活性复筛及HPLC结果显示,菌株XD007、XD114和XD128显著抑制3株病原指示菌,且能产生大量次级代谢产物。【结论】星湖湿地底泥中放线菌资源丰富,筛选到的活性菌株可用于后续药源活性次级代谢产物的分离。     

Hydrogen sulfide attenuates carbon tetrachloride-induced hepatotoxicity, liver cirrhosis and portal hypertension in rats

Background

Hydrogen sulfide (H₂S) displays vasodilative, anti-oxidative, anti-inflammatory and cytoprotective activities. Impaired production of H₂S contributes to the increased intrahepatic resistance in cirrhotic livers. The study aimed to investigate the roles of H₂S in carbon tetrachloride (CCl₄)-induced hepatotoxicity, cirrhosis and portal hypertension.

Methods and Findings

Sodium hydrosulfide (NaHS), a donor of H₂S, and DL-propargylglycine (PAG), an irreversible inhibitor of cystathionine γ-lyase (CSE), were applied to the rats to investigate the effects of H₂S on CCl₄-induced acute hepatotoxicity, cirrhosis and portal hypertension by measuring serum levels of H₂S, hepatic H₂S producing activity and CSE expression, liver function, activity of cytochrome P450 (CYP) 2E1, oxidative and inflammatory parameters, liver fibrosis and portal pressure. CCl₄ significantly reduced serum levels of H₂S, hepatic H₂S production and CSE expression. NaHS attenuated CCl₄-induced acute hepatotoxicity by supplementing exogenous H₂S, which displayed anti-oxidative activities and inhibited the CYP2E1 activity. NaHS protected liver function, attenuated liver fibrosis, inhibited inflammation, and reduced the portal pressure, evidenced by the alterations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), albumin, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and soluble intercellular adhesion molecule (ICAM)-1, liver histology, hepatic hydroxyproline content and α-smooth muscle actin (SMA) expression. PAG showed opposing effects to NaHS on most of the above parameters.

Conclusions

Exogenous H₂S attenuates CCl₄-induced hepatotoxicity, liver cirrhosis and portal hypertension by its multiple functions including anti-oxidation, anti-inflammation, cytoprotection and anti-fibrosis, indicating that targeting H₂S may present a promising approach, particularly for its prophylactic effects, against liver cirrhosis and portal hypertension.     

Discovering motif pairs at interaction sites from protein sequences on a proteome-wide scale

MOTIVATION: Protein-protein interaction, mediated by protein interaction sites, is intrinsic to many functional processes in the cell. In this paper, we propose a novel method to discover patterns in protein interaction sites. We observed from protein interaction networks that there exist a kind of significant substructures called interacting protein group pairs, which exhibit an all-versus-all interaction between the two protein-sets in such a pair. The full-interaction between the pair indicates a common interaction mechanism shared by the proteins in the pair, which can be referred as an interaction type. Motif pairs at the interaction sites of the protein group pairs can be used to represent such interaction type, with each motif derived from the sequences of a protein group by standard motif discovery algorithms. The systematic discovery of all pairs of interacting protein groups from large protein interaction networks is a computationally challenging problem. By a careful and sophisticated problem transformation, the problem is solved using efficient algorithms for mining frequent patterns, a problem extensively studied in data mining. RESULTS: We found 5349 pairs of interacting protein groups from a yeast interaction dataset. The expected value of sequence identity within the groups is only 7.48%, indicating non-homology within these protein groups. We derived 5343 motif pairs from these group pairs, represented in the form of blocks. Comparing our motifs with domains in the BLOCKS and PRINTS databases, we found that our blocks could be mapped to an average of 3.08 correlated blocks in these two databases. The mapped blocks occur 4221 out of total 6794 domains (protein groups) in these two databases. Comparing our motif pairs with iPfam consisting of 3045 interacting domain pairs derived from PDB, we found 47 matches occurring in 105 distinct PDB complexes. Comparing with another putative domain interaction database InterDom, we found 203 matches. AVAILABILITY: http://research.i2r.a-star.edu.sg/BindingMotifPairs/resources. SUPPLEMENTARY INFORMATION: http://research.i2r.a-star.edu.sg/BindingMotifPairs and Bioinformatics online.     

Ubiquitin-specific protease 4 (USP4) targets TRAF2 and TRAF6 for deubiquitination and inhibits TNFα-induced cancer cell migration

TRAF [TNF (tumour necrosis factor)-receptor-associated factor] 2 and 6 are essential adaptor proteins for the NF-κB (nuclear factor κB) signalling pathway, which play important roles in inflammation and immune response. Polyubiquitination of TRAF2 and TRAF6 is critical to their activities and functions in TNFα- and IL (interleukin)-1β-induced NF-κB activation. However, the regulation of TRAF2 and TRAF6 by deubiquitination remains incompletely understood. In the present study, we identified USP (ubiquitin-specific protease) 4 as a novel deubiquitinase targeting TRAF2 and TRAF6 for deubiquitination. We found that USP4 specifically interacts with TRAF2 and TRAF6, but not TRAF3. Moreover, USP4 associates with TRAF6 both in vitro and in vivo, independent of its deubiquitinase activity. The USP domain is responsible for USP4 to interact with TRAF6. Ectopic expression of USP4 inhibits the TRAF2- and TRAF6-stimulated NF-κB reporter gene and negatively regulates the TNFα-induced IκBα (inhibitor of NF-κBα) degradation and NF-κB activation. Knockdown of USP4 significantly increased TNFα-induced cytokine expression. Furthermore, we found that USP4 deubiquitinates both TRAF2 and TRAF6 in vivo and in vitro in a deubiquitinase activity-dependent manner. Importantly, the results of the present study showed that USP4 is a negative regulator of TNFα- and IL-1β-induced cancer cell migration. Taken together, the present study provides a novel insight into the regulation of the NF-κB signalling pathway and uncovers a previously unknown function of USP4 in cancer.     

副溶血性弧菌-霍乱弧菌混合生物被膜形成过程研究

【目的】研究副溶血性弧菌(Vibrioparahaemolyticus,VP)和霍乱弧菌(Vibriocholera,VC)混合生物被膜的形成过程。【方法】在4、8、12、24、36、48、60、72 h测定单独条件下VP、VC及其混合后生物被膜的形成情况,通过结晶紫染色法、平板菌落计数法、测定胞外多糖、胞外蛋白,通过荧光原位杂交(FISH)观察混合生物被膜形成。【结果】虽然形成的混合生物被膜量介于VC和VP之间,但混合生物被膜在形成过程中,成熟期后生物被膜量的变化较小,对环境的抗性增强。混合生物被膜中拥有更多的活菌,混合生物被膜形成过程中胞外蛋白和胞外多糖的变化体现出其可能在对抵御不适应环境中起重要作用,通过FISH可观察到不同时期生物被膜的变化过程。【结论】VC与VP共同形成生物被膜的过程中,混合生物被膜总量虽然减少,但混合生物被膜中拥有更多的活菌,这可能引起更大的危害。研究混合生物被膜形成过程中被膜的变化,可为有害生物被膜的控制提供基础。