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Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels
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Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K(+) Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 alpha-subunits. We found that homomeric channels, formed by monomeric or dimeric alpha-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory subunits. 相似文献
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Salinity tolerance can be attributed to three different mechanisms: Na+ exclusion from the shoot, Na+ tissue tolerance and osmotic tolerance. Although several key ion channels and transporters involved in these processes are known, the variation in expression profiles and the effects of these proteins on Na+ transport in different accessions of the same species are unknown. Here, expression profiles of the genes AtHKT1;1, AtSOS1, AtNHX1 and AtAVP1 are determined in four ecotypes of Arabidopsis thaliana. Not only are these genes differentially regulated between ecotypes, the expression levels of the genes can be linked to the concentration of Na+ in the plant. An inverse relationship was found between AtSOS1 expression in the root and total plant Na+ accumulation, supporting a role for AtSOS1 in Na+ efflux from the plant. Similarly, ecotypes with high expression levels of AtHKT1;1 in the root had lower shoot Na+ concentrations, due to the hypothesized role of AtHKT1;1 in retrieval of Na+ from the transpiration stream. The inverse relationship between shoot Na+ concentration and salinity tolerance typical of most cereal crop plants was not demonstrated, but a positive relationship was found between salt tolerance and levels of AtAVP1 expression, which may be related to tissue tolerance. 相似文献
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人骨髓间充质干细胞向神经细胞分化过程中Notch通路信号分子表达的变化 总被引:6,自引:0,他引:6
本研究探讨Noah信号通路在人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)体外增殖及向神经细胞分化过程中的作用。采集健康自愿者骨髓,体外培养获得hMSCs,取第3代hMSCs,在诱导剂(β-ME,DMSO,BHA)作用下向神经细胞分化。诱导后用免疫细胞化学鉴定神经元特异性烯醇化酶(neuron-specific enolase,NSE)和尼氏体的表达以确定诱导效果:用流式细胞术检测细胞生长周期时相的变化。在诱导前后,用免疫荧光和RT-PCR方法检测Notch通路中Notch1受体蛋白、配体Jagged1(JAG1)、调节蛋白活化相关物早老素1(presenilin 1,PS1)、靶基因hairy and enhancer of split1(HES1)信号分子表达的变化。结果显示:诱导前,处于G0/G1期的hMSCs占58.5%,S+G2/M期的细胞占41.5%;诱导后,G0/G1期细胞比例升高,而S+G2/M期细胞比例下降,NSE阳性细胞率达(77±0.35)%,细胞质中可见深蓝色的块状或颗粒状尼氏体。免疫荧光显示,诱导前后hMSCs内Notch1和JAG1均呈阳性表达,但RT-PCR检测发现诱导后Notch1、JAG1、PSl和HES1 mRNA表达量较诱导前明显降低(均P〈0.05)。结果表明,诱导hMSCs向神经细胞分化能抑制Notch信号分子表达,低水平的Notch信号激活可能有利于神经细胞的分化。 相似文献
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Basil P Hubbard Christine Loh Ana P Gomes Jun Li Quinn Lu Taylor LG Doyle Jeremy S Disch Sean M Armour James L Ellis George P Vlasuk David A Sinclair 《Cell cycle (Georgetown, Tex.)》2013,12(14):2233-2240
SIRT1 is an NAD+-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1. 相似文献
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Enhancement of hepatic microsomal drug oxidation and glucuronidation in rat by 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) 总被引:1,自引:0,他引:1
H Vainio 《Chemico-biological interactions》1974,9(1):7-14
A single dose of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (160 mg/kg i.p.) enhanced the monooxygenase step of drug biotransformation in rat liver. The O-demethylation of p-nitroanisole was especially increased, a peak in activity approximately 5-fold compared with controls being attained in 7 days. On the other hand, there was only a 2-fold increase in aryl hydrocarbon hydroxylase activity.DDT increased the cytochrome P-450 content of the liver, this increase coincided well with that in p-nitroanisole O-demethylation activity.The UDPglucuronosyltransferase activity of liver microsomes was not enhanced by DDT administration, unless the microsomes were pretreated to reveal latent activity prior to assay. After trypsin digestion of microsomes a maximum increase in activity of approximately 3-fold was observed as a result of DDT dosage. The canonic surfactant cetylpyridinium chloride was less active in revealing the latent UDP-glucuronosyltransferase activity, and two other membrane perturbants, the detergent digitonin and phospholipase A, were unable to show enhancement in UDPglucuronosyltransferase as a result of DDT dosage. 相似文献
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甲型H1N1病毒在全世界范围内爆发,引起人们广泛关注,而目前疫苗和新的药物正处于研发阶段,与此同时该病毒神经氨酸酶蛋白序列不断被报道。达菲作为治疗H1N1病毒的药物被患者广泛使用。通过同源性建模的方法比较神经氨酸酶的变异情况,从而预测达菲药物对变异前后的作用效果评价。通过AUTODOCK计算结合能,发现达菲药物与神经氨酸酶的结合能维持在2.4~4.2 kJ/mol范围内,动力学常数最高值达到18.2 mM。证明达菲药物对抑制病毒进入寄主细胞有明显效果。 相似文献
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对一个中国汉族Gilbert综合征遗传家系致病基因突变位点进行鉴定,以期了解该病的分子遗传学基础。首先提取先证者基因组DNA,PCR扩增尿苷二磷酸葡萄糖醛酸转移酶UGT1A1基因的5个外显子,以琼脂糖电泳鉴定PCR产物,纯化后直接测序鉴定。基因扫描显示,与血清胆红素水平密切相关的UGT1A1基因在第1和第5外显子存在纯合突变,而 UGT1A1基因启动子区域和内含子/外显子剪接边界位点序列未检测到突变。进一步对其他家系成员该基因的相应位点进行突变检测,结果显示他们在第1和第5外显子也存在杂合突变,其中还有两个成员在启动子区域检测到(TA)插入突变。对家系成员未抗凝新鲜血液进行生化检测证实了基因突变分析的结果。综合以上结果发现该家系三种突变并存,致病因素为第1和/或第5外显子突变,为显性遗传,两种突变位点纯合导致先证者出现严重胆红素代谢功能障碍。该家系因此成为Gilbert综合征突变位点及其致病机理研究的一个典型临床病例。 相似文献
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目的:探讨代谢酶CYP1A1基因MspI位点多态性与新疆汉族人群肺癌遗传易感性之间的相关性.方法:应用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)技术检测59例新疆汉族肺癌和84例新疆汉族健康人的CYP1A1基因MspI位点多态性分布频率,并分析了CYP1A1基因MspI位点多态性与新疆汉族人群肺癌遗传易感性和患者性别之间的相关性.结果:(1)CYP1A1基因MspI位点3种多态基因型分布频率在两组间比较差异有统计学意义(χ2=6.682,P=0.035),CC基因型在病例组的分布频率显著高于正常对照组.(2)携带突变CC基因型的个体较携带TT基因型的个体患肺癌的危险性增加(OR=3.759.95%CI=1.228-11.494,P=0.035).(3)男女肺癌患者的CYP1A1基因MspI位点基因型及等位基因频率的差异均无显著性(P>0.05).结论:(1)CC突变基因型可能是新疆汉族人群的肺癌易感因素.(2)CYP1A1基因MspI位点多态性可能与新疆汉族肺癌患者的性别无关. 相似文献
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目的:SCCRO/RP42/DCUN1D1是粘膜系统鳞片状细胞癌(SCC)发生时人类基因组3q区域扩增的潜在靶标之一,其蛋白作用机制尚不清楚,本文拟通过表达并大量纯化SCC相关蛋白DCUN1D1用于蛋白结晶以求获得其三维结构。方法:使用人肝脑组织RNA反转录产物为模板扩增出DCUN1D1基因cDNA片断并将其克隆至原核表达载体PGEX-6P-1中,通过IPTG诱导获得大量可溶性表达,再经过GST亲和层析和Sephadex G-200层析柱纯化。结果:获得了纯度95%以上的蛋白,采用悬滴气相扩散法筛选蛋白晶体,获得显微镜下可见的微晶。结论:初步得出DCUN1D1晶体生长条件及范围,为解析DCUN1D1的三维结构并进一步认识其生物功能奠定了基础。 相似文献
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eEF1A1基因克隆、原核分泌表达及融合蛋白纯化 总被引:1,自引:0,他引:1
eEF1A1作为蛋白合成中的重要翻译延伸因子,可与多种功能性蛋白如F-actin、BPOZ-2结合,并在细胞凋亡、蛋白降解方面起重要作用.以往原核基因工程蛋白表达系统大多为包涵体表达的变性分子,需要复性.为了获得eEF1A1原核分泌性可溶性蛋白分子,克隆了人eEF1A1蛋白编码序列(约1 300 bp),并成功构建pET22b-A原核分泌表达重组质粒,转化到大肠杆菌BL21(DE3)菌株,0.4 mmol/L终浓度IPTG诱导,经不同温度下包涵体与胞浆蛋白组分分析,快速明确蛋白表达情况,即诱导4 h后,37℃表达于包涵体组分,在30℃分泌表达至胞浆组分.通过His-Trap亲和层析纯化柱进行线性洗脱,Bradford法测定蛋白浓度高达620 mg/mL,SDS-PAGE分析纯度约为95%,蛋白大小符合50 kD,Western blotting显示目的蛋白能被eEF1A1抗体识别;质谱分析证实重组蛋白为人eEF1A1蛋白分子.为进一步研究其与重要功能性蛋白的相互作用及在细胞凋亡和蛋白降解中的作用奠定基础. 相似文献
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Receptor-like kinases (RLKs) play a prominent role in the interaction between plants and extracellular pathogens. Intriguingly, in the past few years several studies have demonstrated that a number of RLKs influence plant susceptibility to viruses and, in some cases, interact with viral proteins. In this review, we will summarize and discuss recent advances suggesting a direct role for RLKs in plant–virus interactions. 相似文献
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E. G. Shatalova V. I. Loginov E. A. Braga T. P. Kazubskaja M. A. Sudomoina R. L. Blanchard O. O. Favorova 《Molecular Biology》2006,40(2):228-234
Estrogens are critical for breast cancer initiation and development. Sulfotransferase 1A1 (SULT1A1) and UDP-glucuronosyltransferase
1A1 (UGT1A1) conjugate and inactivate both estrogens and their metabolites, thus preventing estrogen-mediated mitosis and
mutagenesis. SULT1A1 and UGT1A1 are both polymorphic, and different alleles encode functionally different allozymes. We hypothesize that low-activity alleles
SULT1A1*2 and UGT1A1*28 are associated with higher risk for breast cancer and more severe breast tumor phenotypes. We performed a case-control
study, which included 119 women of Russian ancestry with breast cancer and 121 age-matched Russian female controls. We used
PCR followed by pyrosequencing to determine the SULT1A1 and UGT1A1 genotypes. Allele UGT1A1*28 was present at a higher frequency than the wild-type UGT1A1*1 allele in breast cancer patients as compared to controls (P = 0.002, OR = 1.79, CI 1.23–2.63). Consistently, the frequency of genotypes that contain allele UGT1A1*28 in the homozygous or the heterozygous state was greater in breast cancer patients as compared with the frequency of the
wild-type UGT1A1*1/*1 genotype (P = 0.003, OR = 4.00, CI 1.49–11.11 and P = 0.014, OR = 2.04, CI 1.14–3.57, respectively). Individuals carrying allele UGT1A1*28 in the homo-or heterozygous state had larger breast tumors (>2 cm) as compared to the group with high-activity genotypes
(P = 0.011, IR = 3.44, CI 1.42–8.36). No association was observed between any of the SULT1A1 genotypes and breast cancer risk or phenotypes. Our data suggest that UGT1A1, but not SULT1A1, genotypes are important for breast cancer risk and phenotype in Russian women.
Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 2, pp. 263–270.
The article was translated by the authors. 相似文献
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植物生长素受体蛋白研究现状 总被引:3,自引:1,他引:2
受体是研究生长素信号传导链的关键环节,因为只有生长素与生长素受体结合以后才会引起后续的级联反应,生长素受体的发现对探索和了解生长素调控机制是极其重要的.目前所发现的生长素结合蛋白(受体)有TIR1和ABP1.扼要的介绍生长素受体TIR1的结构及其与生长素的结合位点,阐述了TIR1在基因水平上的调控和AUX/IAA被泛素化后最终被26S蛋白酶体降解的过程.概述了ABPI的结构、活性位点、性质以及ABP1的作用机理的模型. 相似文献
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Hiroshi Kawauchi Makoto Tsubokawa Atsushi Kanezawa Hiroaki Kitagawa 《Biochemical and biophysical research communications》1980,92(4):1278-1288
Two endorphins have been identified in the teleost pituitary, (chum salmon). Endorphin I is a nonacosa peptide and the primary structure was reported previously. Endorphin II has been elucidated to be a triaconta peptide with the following primary structure: Ac-Tyr-Gly-Gly- Phe-Met-Lys-Ser-Trp-Asn-Glu-Arg-Ser-Gln-Lys-Pro-Leu-Leu-Thr- Leu-Phe-Lys-Asn-Val-Ile-Ile-Lys-Asp-Gly-Gln-Gln-OH. It is evident that these endorphins are highly homologous to each other, but are different molecules, and that endorphin II is much more similar to the mammalian endorphins than endorphin I. 相似文献
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Recent increases in the incidence of obesity and declines in marriage have prompted policymakers to implement policies to mitigate these trends. This paper examines the link between these two outcomes. There are four hypotheses (selection, protection, social obligation and marriage market) that might explain the relationship between marital status transitions and changes in Body Mass Index (BMI). The selection hypothesis suggests that those with a lower BMI are more likely to be selected into marriage. The protection hypothesis states that married adults will have better physical health as a result of the increased social support and reduced incidence of risky behavior among married individuals. The social obligation hypothesis states that those in relationships may eat more regular meals and/or richer and denser foods due to social obligations which may arise because of marriage. Finally, the marriage market hypothesis indicates that when adults are no longer in the marriage market they may not maintain a healthy BMI because doing so is costly and they are in a stable union—or on the other hand, adults may enhance their prospects in the marriage market by losing weight. Taking advantage of longitudinal data and complete marriage histories in the National Longitudinal Survey of Youth 1979, we estimate individual fixed effects models to examine associations between the change in log BMI and the incidence of overweight and obesity, and changes in relationship status controlling for the effects of aging and other respondent characteristics. We find no support for the marriage protection hypothesis. Rather we find evidence supporting the social obligation and marriage market hypotheses—BMI increases for both men and women during marriage and in the course of a cohabiting relationship. Separate analyses by race and ethnicity reveal substantial differences in the response of BMI to relationship status across these groups. 相似文献