Variation in salinity tolerance and shoot sodium accumulation in Arabidopsis ecotypes linked to differences in the natural expression levels of transporters involved in sodium transport

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.     

Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels

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.     

Occurrence of two different endorphins in the salmon pituitary

Two endorphins have been identified in the teleost pituitary, $\text{Oncorhynchus}\text{keta}$ (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.     

三氯乙烷污染环境中脱卤素杆菌的多样性

三氯乙烷(1,1,1-Trichloroethane,TCA)是一种易挥发的氯代烃,密度大于水,曾作为金属清洗剂和高效溶剂在工业上广泛应用.近年来三氯乙烷对环境及人类健康的危害引起人们的关注,其自然分解过程较为困难,在环境中的存在具有一定的持久性.由于不合理的排放和意外泄漏等原因,三氯乙烷已成为地下水中最普遍的污染物之一,严重威胁生态系统和人体健康,是美国环保署(EPA)所确定的优先污染物.研究表明,Peptococcaceae科的脱卤素杆菌属(Dehalobacter)细菌纯培养物或含有Dehalobacter的混合培养物可催化三氯乙烷及1,1,-二氯乙烷还原脱氯,在其降解过程中起关键作用[1 3],并可利用含有Dehalobacter的菌群对三氯乙烷污染场地进行生物治理[4],其中Dehalobacter 16SrRNA基因的特异性探针被用作生物标记物来评价三氯乙烷污染场地的生物降解潜能[4-5].     

Exploring the limits of sequence and structure in a variant betagamma-crystallin domain of the protein absent in melanoma-1 (AIM1)

βγ-Crystallins belong to a superfamily of proteins in prokaryotes and eukaryotes that are based on duplications of a characteristic, highly conserved Greek key motif. Most members of the superfamily in vertebrates are structural proteins of the eye lens that contain four motifs arranged as two structural domains. Absent in melanoma 1 (AIM1), an unusual member of the superfamily whose expression is associated with suppression of malignancy in melanoma, contains 12 βγ-crystallin motifs in six domains. Some of these motifs diverge considerably from the canonical motif sequence. AIM1g1, the first βγ-crystallin domain of AIM1, is the most variant of βγ-crystallin domains currently known. In order to understand the limits of sequence variation on the structure, we report the crystal structure of AIM1g1 at 1.9 Å resolution. Despite having changes in key residues, the domain retains the overall βγ-crystallin fold. The domain also contains an unusual extended surface loop that significantly alters the shape of the domain and its charge profile. This structure illustrates the resilience of the βγ fold to considerable sequence changes and its remarkable ability to adapt for novel functions.     

1981～2005年中国H1N1甲型流感病毒血凝素基因的HA1演变特征

为了解1981~2005年我国H1N1甲型流感病毒血凝素基因的HA1演变特征,选取H1N1甲型流感病毒370株,提取病毒RNA,经逆转录和聚合酶链反应扩增HA1并测序,测定的序列用生物信息软件分析,与GenBank中相关序列比较,并对推导的编码氨基酸序列进行基因特性分析。结果表明:HA1氨基酸的变异表现为抗原决定簇4个区均有变异,Sb区和Ca区变化较大;HA1受体结合位点(RBS)的前壁130环的第134位赖氨酸从1991年起在部分毒株HA1序列上开始缺失,以后缺失株逐步增多,自2000年起测定的所有毒株上该氨基酸全部缺失,同时这些缺失株的第137位氨基酸也全部由苏氨酸替换为丝氨酸;糖基化位点从增多到减少,最后稳定在7个;1981~2004年我国H1N1甲型流感病毒血凝素HA1编码的氨基酸在种系发育树上同年代基本呈现集中分布,与时间和地域无关,2005年毒株分成两个分支在时间上有明显差异。     

eEF1A1基因克隆、原核分泌表达及融合蛋白纯化

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.     

The timing of the rise in U.S. obesity varies with measure of fatness

There are several ways to measure fatness and obesity, each with its own strengths and weaknesses. The primary measure for tracking the prevalence of obesity has historically been body mass index (BMI). This paper compares long-run trends in the prevalence of obesity when obesity is defined using skinfold thickness instead of BMI, using data from the full series of U.S. National Health Examination Surveys. The results indicate that when one uses skinfold thickness rather than BMI to define obesity, the rise in the prevalence of obesity is detectable 10-20 years earlier. This underscores the importance of examining multiple measures of fatness when monitoring or otherwise studying obesity.     

Mipu1基因在内毒素血症小鼠组织中的表达研究

Mipu1是本研究室首次克隆的一个核转录因子,其在内毒素血症中的表达改变情况目前尚不清楚.本研究采用real-time PCR方法检测了Mipu1基因在内毒素血症(12 mg/kg,2 h)小鼠心、肝、肺、脾、脑、肠和骨骼肌7个器官组织中的表达改变.结果发现,LPS(12 mg/kg,2 h)处理可促进小鼠肺和脾组织中Mipu1基因表达增高;但可抑制小鼠心、肝、脑、肠和骨骼肌组织中Mipu1基因的表达.Mipu1基因在内毒素血症小鼠各器官中表达的改变可能与其在内毒素血症中的生物学功能密切相关.     

松嫩平原盐碱土饱和浸提液与土水比1:5浸提液间化学参数的换算关系

采用饱和浸提液与土水比1:5浸提液两种方法分析了松嫩平原94份盐碱土样品的电导率、钠吸附比、主要阳离子(Na+、K+、Ca2+、Mg2+)以及总阳离子浓度等化学参数,并对两种方法测定的盐碱化参数相关性进行了研究.结果表明:饱和浸提液电导率、钠吸附比、总阳离子浓度、Na+离子浓度与土水比1:5浸提液相应参数存在极显著的相关关系,其关系方程可用于松嫩平原盐碱土饱和浸提液与1:5浸提液间化学参数的换算;而K+、Ca2+、Mg2+离子浓度在两种浸提液间不存在相关性.     

粘膜系统鳞状细胞癌(SCC)相关蛋白(DCUN1D1)的表达、纯化和晶体生长

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。     

可溶性细胞间粘附分子在慢性肾脏病中的变化

目的:研究慢性肾病(CKD)患者血清可溶性细胞粘附分子-1(soluble intercellular adhesionmolecule-1,sICAM)的变化与临床意义。方法:用双抗体夹心ELISA方法,对52例CKD患者及20例健康对照人群的sICAM-1水平进行检测分析。52例CKD患者中,其中27例为CRF血液透析患者;25例肾功能正常CKD患者。结果:CKD组患者sICAM-1水平明显高于对照组(105.42±61.95)(P<0.01);肾功能正常CKD组和CKD-CRF组sICAM-1水平均显著高于对照组(P<0.01);CRF组sICAM-1水平明显低于肾功能正常CKD组(P<0.01);但高于对照组(84.80±19.61/164.08±70.66/54.61±5.48)(P<0.01)。结论:sICAM-1水平在慢性肾脏病中明显升高,CRF组病人sICAM-1水平低于CKD肾功能正常患者,提示透析过程中可能有sICAM溢出,吸附并丢失入透析液中(1),或可能是肾纤维化为主的病变使sICAM-1表达下降。     

卡波氏肉瘤相关病毒感染激活Jurkat细胞抗-HIV基因表达的研究

目的:观察Jurkat细胞感染卡波氏肉瘤相关病毒(Kaposi's sarcoma-associated herpes virus,KSHV)后细胞内几条主要的抗HIV作用的基因(RANTES、APOBEC3G、APOBEC3F、MX1、MX2)表达情况。方法:首先用佛波酯(TPA)(20ng/ml)刺激BCBL-1细胞72小时,提取KSHV病毒滤液。然后把含KSHV滤液的RPMI-1640培养Jurkat细胞。24小时后,分别在感染后第3、5天收集细胞,提取细胞总RNA,通过实时定量PCR检测分析相关基因的表达情况。结果:通过对KSHV感染不同时期的细胞抗-HIV基因表达分析,结果显示:与未感染组相比,KSHV感染组的Jurkat细胞内的多条抗HIV-1基因表达上调。感染第3天,RANTES上调123倍,APOBEC3G上调3.12倍,A POBEC3F上调1.18倍,MIX1上调2.75倍,MIX2上调4.35倍,感染第5天RANTES上调11.91倍,MX1上调2.72倍,MIX2上调2.22倍。结论:KSHV感染在一定程度上激活Jurkat细胞抗HIV相关基因的表达。     

Expression of glutamate transporters in human and rat retina and rat optic nerve

l-Glutamate is the major excitatory transmitter in the vertebrate retina and plays a central role in the transmission of the various retinal neurons. Glutamate is removed from the extracellular space by at least five different glutamate transporters. The cellular distribution of these has been studied so far mainly using immunocytochemistry. In the present study non-radioactive in situ hybridisation using complementary RNA probes was applied in order to identify the cell types of rat retina and optic nerve expressing generic GLT1, GLT1 variant (GLT1v or GLT1B), GLAST and EAAC1. The results were compared with immunocytochemical data achieved using affinity-purified antibodies against transporter peptides. In the immunohistochemical studies the human retina was included. The study showed that in the rat retina GLT1v and EAAC1 were coexpressed in various cell types, i.e. photoreceptor, bipolar, horizontal, amacrine, ganglion and Müller cells, whereas GLAST was only detected in Müller cells and astrocytes. In the rat optic nerve GLT1v and EAAC1 were preferentially expressed in oligodendrocytes, whereas GLAST was revealed to be present mainly in astrocytes. Generic GLT1 could not be detected in the retina or optic nerve. The cellular distribution of glutamate transporters (only immunocytochemistry) in the human retina was very similar to that of the rat retina. Remarkable results of our studies were that generic GLT1 was not detectable in the rat (and human) retina and that GLT1v and EAAC1 were demonstrable in most cell types of the retina (including photoreceptor cells and their terminals).     

基质金属蛋白酶及其抑制剂在肝纤维化中的表达变化

目的 观察肝纤维化形成过程中基质金属蛋白酶MMP-1及其抑制剂TIMP-1的表达变化,从细胞外基质降解代谢的角度研究四氯化碳(CCl4)中毒性肝纤维化发生的机制.方法 雄性Wistar大鼠20只,分为正常组和肝纤维化模型组.肝纤维化组采用CCl4、饮酒、高脂低蛋白饮食等复合病因刺激制备肝纤维化动物模型,造模时间为8周.实验结束后测定肝脏指数、血清透明质酸(HA)、谷丙转氨酶(ALT)及尿羟脯氨酸(HYP)排出量,光镜下观察肝组织纤维化程度,并用免疫组化SABC法检测肝组织中Ⅰ、Ⅲ型胶原蛋白及MMP-1、TIMP-1的表达,同时用荧光实时定量PCR(RT-PCR)的方法检测肝组织中MMP-1、TIMP-1 mRNA的表达.结果 与正常对照组比较,肝纤维化模型组大鼠肝脏指数、血清HA及ALT显著增高,尿羟脯氨酸的排出量明显增加,病理组织学检查发现肝组织内纤维结缔组织增生明显,有假小叶形成;免疫组化的结果显示肝组织内Ⅰ、Ⅲ型胶原蛋白、MMP-1及TIMP-1的表达较正常组显著增加.结论 肝组织中MMP-1及TIMP-1的表达变化可能是导致肝纤维化的重要机制之一.     

一个中国Gilbert综合征家系的遗传学分析

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。