超抗原TSST-1之靶TCRVβHV4序列的特征分析

首先对人和小鼠的TCRVβ的氨基酸序列进行多序列对准,就Vβ之HV4片段进行比较,分析与超抗原TSST-1结合的四种Vβ（小鼠Vβ3、Vβ15、Vβ17和人Vβ2）之HV4序列内是否存在特定的氨基酸残基排列主型。结果发现:小鼠Vβ3和Vβ17的HV4具有特异的RFSAXCXSNS主型,而小鼠Vβ15和人β2的HV4则含独特的KFXIXH主型。提示:与TSST-1结合的四种Vβ所对应的T细胞识别表位可能不止一个。     

高原鼠兔血红素氧合酶1的基因克隆及序列分析

 [摘 要] 目的:克隆高原鼠兔血红素氧合酶1（heme oxygenase-1,HO-1）基因cDNA的全长序列,并分析其序列特征,为进一步揭示高原鼠兔低氧适应的分子机制提供有益参考。方法:从高原鼠兔肝组织中提取总RNA,利用逆转录聚合酶链反应（RT-PCR）技术和cDNA末端快速克隆（RACE）技术扩增出HO-1基因cDNA全长序列并进行测序,测序结果采用生物信息学的方法进行分析。结果:克隆所得鼠兔HO-1基因cDNA全长片段大小为1 466 bp,与预期一致,编码区长度为873   bp,编码290个氨基酸（GenBank登录号为：JX035934）;序列分析结果显示,核苷酸序列和推测的氨基酸序列相似性比较,与兔、人、牛、小鼠、大鼠、猪和马的HO-1核苷酸序列的同源性分别为89%、87%、85%、79%、84%、85%和85%,与兔、人、牛、小鼠、大鼠、猪和马的HO-1氨基酸序列的同源性分别为89%、85%、84%、80%、79%、82%和67%,显示出高度保守性。构建的基于氨基酸序列的分子系统进化树聚类结果表明,高原鼠兔与兔的进化距离最近。结论: 本实验首次成功克隆出高原鼠兔HO-1基因cDNA全长序列,为从低氧细胞保护角度,进一步探讨青藏高原土著物种适应高原的分子生物学机制研究提供实验依据。     

一个人类α-甘露糖苷酶全长cDNA的分子克隆

用我们以往克隆到的1358bp6A8cDNA片段作探针,籍southernblot从一个人扁桃体细胞λgtllCDNA文库筛选,再用RACE方法,克隆到了一个长3300bp的6A8全长CDNA。此CDNA内有一长3188hp（57～3245hp）的开放阅读框架,编码1064个氨基酸的蛋白质。此蛋白质的氨基酸序列与大鼠一个ER－α－甘露糖苦酶（1040个氨基酸）的相同性和相似性高达78％和82％,与一个酵母－α－甘露糖着酶（1083个氨基酸）的相同性和相似性亦达33％和47％。另外,值得注意的是其260～432位氨基酸序列与人类、猪、大鼠和小鼠的多种α－甘露糖甘醇的相应氨基酸序列的相同性均高于20％,提示这段保守序列与α－甘露糖着酶的功能可能有关。     

ANALYSIS OF MOTIF IN TCR Vβ HV4 SEQUENCES BINDING SUPERAN…

首先对４１种人和小鼠的Ｔ细胞受体β链可变基因编码肽段（Ｖβ）的氨基酸序列进行序列对准，就Ｖβ之第四高变区（ＨＶ４）片段进行比较，分析与超抗病毒素休克综合征毒素－１（ＴＳＳＴ－１）结合的四种Ｖβ（小鼠Ｖβ３、Ｖβ１５、Ｖβ１７和人Ｖβ２）之ＨＶ４序列内是否存在特定的氨基酸残基排列模式。结果发现：小鼠Ｖβ３和Ｖβ１７的ＨＶ４具有特异的ＲＦＳＡＸＣＸＳＮＳ模式，而小鼠Ｖβ１５和３人Ｖβ２的ＨＶ４则含独特     

超抗原TSST—1之靶PCRVβHV4序列的特征分析

首先对人和小鼠的ＴＣＲＶβ的氨基酸序列进行多序列对准，就Ｖβ之ＨＶ４片段进行比较，分析与超抗原ＴＳＳＴ－１结合的四种Ｖβ之ＨＶ４序列内是否存在特定的氨基酸残基排列主型。     

人、大鼠、豚鼠、猪、狗和牛胰腺生长抑素免疫反应细胞形态和分布的比较研究

用免疫组织化学ＡＢＣ法，观察了人、大鼠、豚鼠、猪、狗和牛胰腺体部生长抑素样免疫反应（ＳＳＬＩ）细胞的形态和分布。结果显示：ＳＳＬＩ细胞在人和豚鼠胰腺散在分布于整个胰岛，细胞较大，呈卵圆形、圆形或梨形，常可见较长的胞质突起。在豚鼠外分泌部的腺泡，偶见散在的圆形或卵圆形ＳＳＬＩ细胞镶嵌于腺泡细胞间。在大鼠、猪和牛，ＳＳＬＩ细胞集中分布于胰岛的周边部，较少看到胞质突起。在狗胰腺，ＳＳＬＩ细胞集中分布在胰岛中央，周边部很少见到，细胞较小；除胞质外，胞核也含有ＳＳＬＩ阳性物质。在细胞数量上，以豚鼠和狗的单位胰腺组织面积内细胞最多，牛次之，人、大鼠和猪则较少。在ＳＳＬＩ细胞所占胰岛面积的百分比上，狗胰腺的ＳＳＬＩ细胞最高，为３３％左右。豚鼠次之，为１８％；人和牛分别为１１％和９％，大鼠和猪都在５％左右。哺乳动物胰腺ＳＳＬＩ细胞形态、分布及数量上的差别，说明ＳＳ在不同种属动物胰腺中的作用方式、作用强度和范围有所不同。     

大鼠不同发育阶段β-防御素-2基因

目的:探讨大鼠β-防御素-2基因在肺组织表达的发育调控。方法:根据Genbank大鼠β-防御素-2的cDNA序列,设计一对特异引物。采用RT-PCR技术在大鼠不同发育阶段的肺组织总RNA中,扩增其特异cDNA片段,并进行DNA序列测定。借助Genbank中BLAST程序,将从该cDNA序列推导的氨基酸序列与人的hBD-2和大鼠的rBD-1做相似性分析。结果:从足月胎鼠、出生8h和4d以及成年大鼠相同重量肺组织所提取的总RNA中,均扩增出一条密度相同、长度约为220bp的cDNA片段。在所推导氨基酸序列中与hBD-2氨基酸相同率为48.6%(17/35),与rBD-1氨基酸残基的相同率为31%(11/35)。其成熟分子链长35个氨基酸残基,含β-防御素共有的且排列次序相同的6个典型的半胱氨酸和其它保守氨基酸残基。结论:本实验表明不同发育阶段大鼠肺组织均表达β-防御素-2mRNA,提示该分子可能是肺组织天然抵抗微生物感染的一个重要分子基础。     

一个与编码大鼠ERα—甘露糖苷酶同源的人cDNA分子克隆

用抗人Ｂ细胞和Ｔ细胞共同分化抗原６Ａ８单克隆抗体从人扁桃体细胞λｇｔ１１ｃＤＮＡ文库克隆到１个长１３５８ｂｐ的ｃＤＮＡ。此ｃＤＮＡ内有一个长１２７８ｂｐ（２６～１３０３ｂｐ）的开放阅读框架，编码４２５个氨基酸的蛋白质。此蛋白质氨基酸序列中第１５７位到２２３位为疏水跨膜区。蛋白质的氨基酸序列与大鼠ＥＲα－甘露糖苷酶（１０４０个氨基酸）的６３２～１０３８位氨基酸序列间的相同性和相似性高达８２．０４５％和８８％。它与酵母Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｖｉｓｉａｅα－甘露糖苷酶（１０８３个氨基酸）的６５９～１０８３位氨基酸序列间的相同性和相似性也有３２．５％和５１．７５％。前者１６３～２７７位间与后者８３２～９４６位间１１５个氨基酸序列间的相同性和相似性达到５７％和７４％。由于６Ａ８ｃＤＮＡ核苷酸顺序未见于国外基因库资料，故已为ＮＩＨＧｅｎＢａｎｋ接受登记，接受号为Ｕ３７２４８。     

不同年龄小鼠房室结的光镜观察和定量分析

目的：研究小鼠房室结组织结构和年龄变化。方法：用石蜡切片，HE和Masson染色，光镜观测，用图像分析仪分析房室结区的组织结构特点。结果：小鼠房室结位于中心纤维体前下方，房室隔右侧的心内膜下，主要由P细胞和T细胞构成。3例有副房室结存在。6天鼠，2月鼠和10－12月鼠P细胞的面积分别是124．50，190．00，115．10μm^2;T细胞的面积分别为113．20，136．50和114．80μm^2。胶原占房室结的面积分别是12．85％，25．54％和23．44％。结论：小鼠房室结的P细胞和T细胞随年龄的增长而增大。但10－12月期接近6天期大小。     

利用同源序列确定人GIPC2核心启动子和寻找可能的顺式作用元件

目的利用同源序列确定人GIPC2核心启动子区和寻找可能存在的基因调控区域。方法 1)利用Vista比对不同物种GIPC2基因编码区和非编码区序列,得到GIPC2同源序列;2)分别构建插入人和大鼠GIPC2翻译起始位点ATG上游不同长度截短片段的重组质粒,转染人HEK293T细胞和大鼠PC12细胞;3)根据报告基因荧光强度推断人和大鼠GIPC2的核心启动子区;4)将人GIPC2内含子区同源序列置于核心启动子前面,构建重组质粒,转染HEK293T,HT-29和PC12细胞,检测启动子活性。结果人GIPC2核心启动子区确定为+32至+190序列区域;人和大鼠GIPC2 ATG上游一段同源序列有抑制GIPC2启动子活性的功能;人和大鼠GIPC2第一个内含子内的同源序列有促进启动子活性的功能。结论利用同源序列方法确定了人GIPC2基因的核心启动子区,并且找到能抑制和促进GIPC2启动子活性的可能的顺式作用元件。     

Effects of hypothyroidism and hyperthyroidism on thermogenic responses to selective and nonselective beta-adrenergic agonists in rats

Oxygen consumption (VO2) and mitochondrial guanosine diphosphate (GDP) binding of interscapular brown adipose tissue (BAT) were measured in hypothyroid, hyperthyroid and euthyroid rats after stimulations with selective and nonselective beta-adrenoceptor agonists: BRL 35135A (BRL) and Isoprenaline (ISO). Resting VO2, VO2 increment and mitochondrial GDP binding after beta-adrenergic stimulations were lower in hypothyroid rats than in the euthyroid group. The reduced responses were more marked for ISO than for BRL. Restion VO2 and VO2 increment after beta-adrenergic stimulations were higher in hyperthyroid rats than in the eurthyroid group; the increment was more marked for BRL than for ISO. In hyperthyroidism, mitochondrial GDP binding after BRL and after ISO was in the same magnitude; it was higher in the hyperthyroid than in the euthyroid group after BRL but not after ISO. The different thermogenic responses after ISO and BRL stimulations suggest that BRL is acting on a beta-adrenoceptor differing from the beta-1 and beta-2 adrenoceptors responsible for the effects of ISO. Activation of thermogenesis via the beta-3 adrenoceptor seems to be less dependent on the permissive levels of thyroid hormones than activation via beta-1 and/or beta-2 adrenoceptors. The beta-3 adrenoceptor may be more sensitive to increased levels of thyroid hormones.     

The granzyme B gene is highly polymorphic in wild mice but essentially invariant in common inbred laboratory strains

Granzyme B is a 247 amino acid pro-apoptotic protease secreted by effector lymphocytes for the purpose of killing virus-infected cells. While the capacity of granzyme B to potently induce caspase-dependent apoptosis has long been recognized, it has only recently been found that human and mouse granzyme B activate overlapping but distinct apoptotic pathways. To investigate a possible evolutionary basis for this observation, we sequenced the exons and flanking intronic sequences of the mouse Gzmb gene from a variety of inbred laboratory strains and wild mice. The sequences of 12/13 inbred strains encoded identical proteins, the exception being DBA/2, whose sequence varied at two amino acids. By contrast with the laboratory strains, there was extensive polymorphism in the Gzmb gene of 54 wild mice and 28 wild-derived inbred mice examined, resulting in 2-18 amino acid differences in the predicted proteins, a discrepancy rate of up to 7.3%. Many of these amino acid variations were found in rat and/or human granzyme B. The granzyme B allotype of inbred laboratory strains could be identified in only one of three geographically dispersed clans of wild mice and was absent from all 28 wild-derived inbred strains. The Gzmb gene of Mus musculus castaneus, a close relative of laboratory mice, encoded six amino acid differences compared with the laboratory strains, all of which were also found in corresponding positions in the granzyme B molecules of wild mice. Unlike the protease, the extended granzyme B recognition and cleavage site in Bid, a key pro-apoptotic substrate, was invariant.     

Respiratory-burst stimulants desensitize beta-2 adrenoceptors on human polymorphonuclear leukocytes

We investigated the effect of respiratory-burst stimulants on beta-2 adrenoceptors in human polymorphonuclear leukocytes (PMNL). Pre-incubation of PMNL with these substances did not affect the number or affinity of the receptors but desensitized them, as shown by the "right-shift" in (-)-isoproterenol competition isotherms. H-7, an established protein kinase C inhibitor, and nimesulide, a new putative inhibitor of this enzyme, blunted both superoxide anion production and beta-2 adrenoceptor desensitization. A positive correlation was found between superoxide anion generation and the "right-shift" in isoproterenol competition isotherm (r = 0.92; p less than 0.01). Desensitization of beta-2 adrenoceptors was not due to superoxide anions per se since incubation of PMNL with superoxide anion scavengers (superoxide dismutase and catalase) did not modify the results.     

Cellular and subcellular localization of alpha-1 adrenoceptors in the rat visual cortex

Noradrenaline is thought to play modulatory roles in a number of physiological, behavioral, and cellular processes. Although many of these modulatory effects are mediated through alpha-1 adrenoceptors, basic knowledge of the cellular and subcellular distributions of these receptors is limited. We investigated the laminar distribution pattern of alpha-1 adrenoceptors in rat visual cortex, using immunohistochemistry at both light and electron microscopic levels. Affinity-purified anti-alpha-1 antibody was confirmed to react only with a single band of about 70-80 kDa in total proteins prepared from rat visual cortex. Alpha-1 adrenoceptors were widely distributed though all cortical layers, but relatively high in density in layers I, II/III, and V. Immunoreactivity was observed in both neuronal perikarya and processes including apical dendrites. In double-labeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-glial fibrillary acidic protein, anti-glutamic acid decarboxylase 65/67, anti-2-3-cyclic nucleotide 3-phosphodiesterase, and anti-tyrosine hydroxylase antibodies, alpha-1 adrenoceptors were found mainly in dendrites and somata of microtubule-associated protein 2-immunopositive neurons. About 20% of alpha-1 adrenoceptors were in GABAergic neurons. A small number of alpha-1 adrenoceptors were also distributed in axons of excitatory neurons, astrocytes, oligodendrocytes and noradrenergic fibers. Using an immunoelectron microscopic technique, numerous regions of alpha-1 adrenoceptor immunoreactivity were found in cell somata, on membranes of dendrites, and in postsynaptic regions. Moreover, a small number of immunoreaction products were also detected in axons and presynaptic sites. These findings provide the first quantitative evidence regarding the cellular and subcellular localization of alpha-1 adrenoceptor immunoreactivity in visual cortex. Moreover, the ultrastructural distribution of alpha-1 adrenoceptor immunoreactivity suggests that alpha-1 adrenoceptors are transported mainly into dendrites and that they exert effects at postsynaptic sites of neurons.     

Antibodies to beta 1 and beta 2 adrenoreceptors in Chagas'' disease.

Evidence accumulated over the last decade concerning human and experimental models suggests that an immunopathological mechanism may be involved in the pathogenesis of chronic Chagas' disease. In this paper we demonstrate the existence of two different circulating IgG in chagasic patients which bind with myocardial beta 1 and spleen cell beta 2 adrenoceptors, acting as non-competitive inhibitors. Both chagasic IgG against beta 1 and beta 2 adrenoceptor increased intracellular levels of cAMP that could be blocked by specific beta 1 and beta 2 adrenoceptor antagonists. The specificity for beta 1 and beta 2 adrenoceptors and the independence of other tissue reactive antibodies was demonstrated by IgG absorption with turkey red blood cell (TRBC), human lymphocytes (HL) or guinea pig red blood cells (GPRBC). The F(ab')2 fraction acted similarly. This supports the specificity of beta 1 and beta 2 adrenoceptors to the chagasic IgG and the independence of the other tissue reactive antibodies, such as EVI system. The probable pathogenic role of both beta 1 and beta 2 adrenergic chagasic antibody is discussed.     

Gene 8 of influenza virus: Sequences of cDNA transcribed from the 3′ ends of viral RNA of influenza A and B strains

Nucleotide sequences of cDNA transcribed from the Tends of RNA segment 8 of influenza strains A/PR/8/34 (HONI), A/RI/5-/57 (H2N2), and B/Lee/40 have been obtained by the dideoxy method, with depurination analysis of specific dideoxy-terminated products used to partially confirm the sequences. In the first 80 nucleotides there are 3 differences when the sequences of segment 8 from PR8 and R15^? are compared, whereas a comparison of the first 130 nucleotides of cDNA transcribed from R15^? and B/Lee segment 8 shows no similarities either in the nucleotide sequences or predicted amino acid sequences of the “nonstructural” proteins coded by segment 8.     

Molecular characterization of the genomic S RNA segment from lymphocytic choriomeningitis virus

We have used cDNA clones derived from the genomic S RNA segment of lymphocytic choriomeningitis virus (LCMV), Armstrong strain, as hybridization probes to monitor virus gene expression during acute infections. Our results with strand-specific probes confirm the ambisense character of the LCMV S RNA segment and document the presence of both genomic sense and genomic complementary sense RNA species over the time course of infection. We have used nucleotide sequence information to predict primary amino acid sequences for the major viral structural proteins, nucleoprotein (NP) and glycoprotein (GP-C). Antibodies raised against synthetic peptides derived from these predicted protein sequences have indicated that the gene order for the S segment is 3' NP----5' GP-C and provided direct demonstration that the GP-1 portion of the GP-C precursor is encoded nearest the 5' end of the S segment. Comparison of the predicted amino acid sequences for NP and GP-C between the Armstrong CA-1371 strain and the WE strain shows over 90% amino acid identity. This suggests that significant differences described for the pathogenic potential of the Arm and WE strains in C3H mice reside in one or a very few critical amino acid changes.     

Species variations in cDNA sequence and exon splicing patterns in the extensible I-band region of cardiac titin: relation to passive tension

Titin is believed to play a major role in passive tension development in cardiac muscle. The cDNA sequence of cardiac titin in the I-band sarcomeric region was determined for several mammalian species. Contiguous sequences of 3749, 12,230, 6602, and 11,850 base pairs have been obtained for the rat N2B, rat N2BA, dog N2B, and dog N2BA isoforms respectively. The length of the PEVK region of the N2B isoform did not correlate with rest tension properties since the only species showing an altered length was the dog that expressed a shorter form. No differences were found between the N2B PEVK lengths in ventricular and atrial muscle. New N2BA splicing pathways in the first tandem Ig region were found in human and dog cardiac muscle. Most of the rat and dog sequences were 85–95% identical with the reported human sequence. However, the N2B unique amino acid sequences of rat and dog were only 51 and 67% identical to human. The rat N2B unique sequence was 526 amino acids in length compared to 572 in human. The difference in length was due to deletion of amino acid segments from six different regions of the N2B unique domain. Patterns of PEVK exon expression were also much different in the dog, human, and rat. Six separate dog N2BA PEVK clones were sequenced, and all had different exon splice combinations yielding PEVK lengths ranging from 703 to 900 amino acids. In contrast a rat N2BA clone had a PEVK length of 525 amino acids, while a human clone had an 908 amino acid PEVK segment. Thus, in addition to the higher proportion of the shorter N2B isoform found in rat compared with dog cardiac muscle observed previously, shorter N2B unique and N2BA PEVK segments may also contribute to the greater passive tension in cardiac muscle from rats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Deletion of the α2A/α2C‐adrenoceptors accelerates cutaneous wound healing in mice

The α2‐adrenoceptors regulate the sympathetic nervous system, controlling presynaptic catecholamine release. However, the role of the α2‐adrenoceptors in cutaneous wound healing is poorly understood. Mice lacking both the α2A/α2C‐adrenoceptors were used to evaluate the participation of the α2‐adrenoceptor during cutaneous wound healing. A full‐thickness excisional lesion was performed on the dorsal skin of the α2A/α2C‐adrenoceptor knockout and wild‐type mice. Seven or fourteen days later, the animals were euthanized and the lesions were formalin‐fixed and paraffin‐embedded or frozen. Murine skin fibroblasts were also isolated from α2A/α2C‐adrenoceptor knockout and wild‐type mice, and fibroblast activity was evaluated. The in vivo study demonstrated that α2A/α2C‐adrenoceptor depletion accelerated wound contraction and re‐epithelialization. A reduction in the number of neutrophils and macrophages was observed in the α2A/α2C‐adrenoceptor knockout mice compared with wild‐type mice. In addition, α2A/α2C‐adrenoceptor depletion enhanced the levels of nitrite and hydroxyproline, and the protein expression of transforming growth factor‐β and vascular endothelial growth factor. Furthermore, α2A/α2C‐adrenoceptor depletion accelerated blood vessel formation and myofibroblast differentiation. The in vitro study demonstrated that skin fibroblasts isolated from α2A/α2C‐adrenoceptor knockout mice exhibited enhanced cell migration, α‐smooth muscle actin _protein expression and collagen deposition compared with wild‐type skin fibroblasts. In conclusion, α2A/α2C‐adrenoceptor deletion accelerates cutaneous wound healing in mice.