Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain

A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K⁺ current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure–function relation of KCNQ1 channel and treatments of cardiac channelopathies.     

Crystal structures of 26 kDa Clonorchis sinensis glutathione S-transferase reveal zinc binding and putative metal binding

The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0 Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST–D26H structure showed that four histidine residues – His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule – coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.     

Identification of Rice Ethylene-Response Mutants and Characterization of MHZ71OsEIN2 in Distinct Ethylene Response and Yield Trait Regulation

Ethylene plays essential roles in adaptive growth of rice plants in water-saturating environment; how- ever, ethylene signaling pathway in rice is largely unclear. In this study, we report identification and characterization of ethylene-response mutants based on the specific ethylene-response phenotypes of etiolated rice seedlings, includ- ing ethylene-inhibited root growth and ethylene-promoted coleoptile elongation, which is different from the ethylene triple-response phenotype in Arabidopsis. We establish an efficient system for screening and a set of rice mutants have been identified. Genetic analysis reveals that these mutants form eight complementation groups. All the mutants show insensitivity or reduced sensitivity to ethylene in root growth but exhibit differential responses in cole0ptile growth. One mutant group mhz7 has insensitivity to ethylene in both root and coleoptile growth. We identified the corresponding gene by a map-based cloning method. MHZ7 encodes a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. Upon ethylene treatment, etiolated MHZ7-overexpressing seedlings exhibit enhanced coleoptiie elongation, increased mesocotyl growth and extremely twisted short roots, featuring enhanced ethylene- response phenotypes in rice. Grain length was promoted in MHZ7-transgenic plants and 1000-grain weight was reduced in mhz7 mutants. Leaf senescent process was also affected by MHZ7 expression. Manipulation of ethylene signaling may improve adaptive growth and yield-related traits in rice.     

Evidence of colorectal cancer risk associated variant Lys25Ser in the proximity of human bone morphogenetic protein 2

Colorectal cancer (CRC) is the third most prevalent cancer and fourth leading cause of cancer-related deaths globally. It has been shown that the nsSNP variants play an important role in diseases, however it remained unclear how these variants are associated with the disease. Recently, several CRC risk associated SNPs have been discovered, however rs961253 (Lys25Arg at 20p12.3) located in the proximity of bone morphogenetic protein 2 (Bmp2) and fermitin family homolog 1 Fermt1 genes have been reported to be highly associated with the CRC risk. Here we provide evidence for the first time in silico biological functional and structural implications of non-synonymous (nsSNPs) CRC disease-associated variant Lys25Arg via molecular dynamic (MD) simulation. Protein structural analysis was performed with a particular variant allele (A/C, Lys25Arg) and compared with the predicted native protein structure. Our results showed that this nsSNP will cause changes in the protein structure and as a result is associated with the disease. In addition to the native and mutant 3D structures of CRC associated risk allele protein domain (CRAPD), they were also analyzed using solvent accessibility models for further protein stability confirmation. Taken together, this study confirmed that this variant has functional effect and structural impact on the CRAPD and may play an important role in CRC disease progression; hence it could be a reasonable approach for studying the effect of other deleterious variants in future studies.     

Molecular dynamics investigation on the poor sensitivity of A171T mutant NEDD8-activating enzyme (NAE) for MLN4924

MLN4924 is an adenosine sulfamate analog that generates the inhibitory NEDD8-MLN4924 covalent complex. A single nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3 reduces the enzyme’s sensitivity for MLN4924. Our molecular dynamics simulation study revealed that A171T transition brought remarkable conformational changes in enzyme structure (open ATP binding pocket), which reduced the interaction between MLN4924 and ATP binding pocket while wild form completely covered the MLN4924. A total difference of ?49.75?kJ/mol was noticed in interaction energy (electrostatic and van der Waals) during simulation between mutant and wild form with MLN4924. Superimposition of final 20?ns mutant structure with reference structure showed significant change in native binding position as compared to wild form. Results were found in coherence with the recently reported in vitro studies which states that A171T transition leads to change in ATP binding pocket structure.     

单核细胞增生性李斯特菌prfA基因缺失株的构建及其生物学特性

【目的】单核细胞增生性李斯特菌(Lm)是人兽共患李斯特菌病的病原菌,其致病性与调控因子PrfA蛋白作用下毒力基因的表达有着密切关系,本文初步探讨了PrfA蛋白对细菌毒力因子的调控作用。【方法】利用同源重组技术对血清型分别为1/2a和4b的LM4、F4636进行prfA基因的敲除,并构建其回复突变株,对获得的突变株LM4ΔprfA、F4636ΔprfA进行生物学特性研究。【结果】实验结果表明:两株缺失株的溶血活性丧失、回复突变株的溶血活性得到恢复,突变株还丧失磷脂酶活性,黏附和侵袭特性显著下降(P<0.05),对BALB/c小鼠的半数致死剂量提高了105个数量级。【结论】由此表明,PrfA蛋白对hly、plcB、inl家族基因的表达及细菌毒力具有重要的调控作用。prfA基因缺失株的构建为进一步研究PrfA蛋白的调控功能提供了材料,为研究其在Lm致病性中的作用奠定了基础。     

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine‐independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non‐receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3‐positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL‐positive leukaemia. Taken together, our results support the notion that mutant CBL‐expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Fatty Acid Compositions of Corynebacterium simplex Grown on 1-Alkenes

The utilization of 1-alkenes by Corynebacterium simplex ATCC 6946 was studied with respect to the characteristic fatty acid profiles resulting from the growth at the expense of these substrates.

It was indicated that the synthetic pathways of the cellular fatty acids in Corynebact. simplex grown on various n-alkanes or 1-alkenes changed markedly according to the chain lengths of the substrates. From shorter chain hydrocarbons (C₁₂, C₁₄) the fatty acids were found to be synthesized mainly via de novo synthesis pathway in a similar manner to those from glucose, while chain elongation and intact incorporation occurred to a very small extent. On the other hand, an intact incorporation mechanism was preferential in the cells grown on longer ones (C₁₆, C₁₈). When n-pentadecane or 1-pentadecene was used as the substrate, these three mechanisms seemed to operate simultaneously.     

Fatty Acids Associated with the Cell Wall in Film Strains of Saccharomyces

Fatty acid contents were estimated in the cell wall of Saccharomyces. The fatty acids responsible for cell wall hydrophobicity were classified by ease of extraction to ‘readily extractable’ and ‘bound’ acids. The readily extractable fatty acids were easily extracted with pentane and chloroform-methanol. The fatty acids extracted with chloroform-methanol were quite effective for cell wall hydrophobicity, but the fatty acids extracted with pentane were not. The bound fatty acids comprised in the phospholipids phosphatidylethanolamine and phosphatidylserine, which were rigidly associated with the cell wall. These phospholipids were not extractable until they were released from the cell wall by pronase. Chloroform-methanol extraction caused a reduction in cell wall phospholipid content, particularly after treatment with pronase. The fatty acid content of the resultant cell wall was lowered to below 7% of initial content. Phospholipids contained more saturated fatty acid than readily extractable lipids. Phospholipids greatly contributed to cell wall hydrophobicity of various film strains of Saccharomyces.