应用细菌双杂交系统技术寻找与人PCIA1基因相互作用的基因

目的探寻与新近发现的人类交叉免疫反应抗原（homo sapiens cross-immune reaction antigen）基因PCIA1相互作用的基因,为其功能研究提供线索。方法应用Stratagene公司的细菌双杂交系统和人胚肾cDNA文库,构建诱饵质粒pBT-PCIA1,与靶质粒pTRG-cDNA文库大规模地共转化报告菌株,筛选并分离具有相互作用的pTRG-cDNA阳性克隆,经DNA序列分析和生物信息学确定靶基因。结果发现7种靶蛋白基因,其中3种基因功能尚不清楚,其余4种具有重要生理功能,突变或表达异常都会导致严重疾病;α-辅肌动蛋白-4（actinin,alpha4,ACTN4）、鞘脂激活蛋白原（prosaposin,PSAP）或真核翻译起始因子（eukary-otic translation initiation factor 3,subunit 10 theta,EIF3S10）过表达都会促进肿瘤发生和演进。结论细菌双杂交系统技术探测蛋白质之间的相互作用,是一种提供新基因功能研究信息的有效方法;PCIA1基因表达与肿瘤形成、侵袭和转移密切相关。     

HMGA蛋白在恶性外周神经鞘膜肿瘤和滑膜肉瘤中的表达

恶性外周神经鞘膜肿瘤（MPNST）是一种比较常见的起源于外周神经或向神经鞘膜方向分化的恶性肿瘤，该肿瘤无论在组织形态学上还是免疫组化上需要和梭形细胞肉瘤，特别是滑膜肉瘤鉴别。HMG是一种低分子量的非组蛋白，功能是作为转录调控蛋白，结合DNA和修饰染色质构型。HMGI家族成员包括HMGA1和HMGA2，HMGA基因失调可见于多种良性间叶性肿瘤，动物实验发现它们在间叶组织和肿瘤发生中起重要作用。     

光镜水平下Fabry病溶酶体贮积物的检测和定性

Ｆａｂｒｙ病是由于细胞溶酶体中α 半乳糖苷酶遗传性缺失 ,导致该酶的底物 ,神经鞘脂类化合物的正常降解受阻 ,神经鞘脂类化合物在溶酶体中贮积而导致的疾病[1,2 ] 。神经鞘脂类化合物由酰基鞘氨醇及第一碳位上的一个或多个糖残基组成。α 半乳糖苷酶是一种可水解神经鞘脂类化合物中酰基鞘氨醇三巳糖苷脂末端α 半乳糖残基的外糖苷酶 ,其功能缺失引起酰基鞘氨醇三巳糖苷脂在血管内皮细胞、纤维母细胞和汗腺细胞等细胞的溶酶体中堆积 ,从而导致皮肤和黏膜角质瘤、肢端疼痛和感觉异常、少汗、心血管及肾功能不全等多种临床表现[3 ] 。从遗传…     

重组人血小板源生长因子的药学研究进展

血小板源生长因子（platelet-derived growth factor， PDGF）是一种通常存贮于血小板α颗粒中的碱性蛋白质，由血小板趋化到受损部位的巨噬细胞，受损血管处的平滑肌细胞以及受损部位的血管内皮细胞等多种细胞所分泌。自从发现 PDGF 后，人们已成功地纯化了人、猪、牛、羊等的PDGF，并对其理化性质进行了大量的研究。PDGF 是一种热稳定的阳离子型糖蛋白，分子量为28~35 kD，是由分子量分别为13~14 kD 和16~17 kD 的两个亚基通过二硫键连接组成。PDGF 的等电点为9.8~10.2，具有耐高温（100℃）、耐酸（pH 2.5）以及耐受各种解离剂（4 mol/L 盐酸胍、6 mol/L 尿素、2%SDS）的特点。凝胶层析发现，人PDGF 在体内有两种形式，即 PDGF-I（分子量为31 kD）和 PDGF-II（分子量为28 kD），两者在氨基酸及以共价键相连的碳水化合物的组成上有所不同，但其致丝裂活性相似[1]。     

鞘脂代谢与心血管疾病关系的研究进展

鞘脂作为一种生物活性脂质,可参与生长、凋亡等多种重要生理过程的信号转导,且在包括心血管疾病在内的多种疾病状态中表现出了异常水平,提示鞘脂代谢参与心血管疾病的发生发展.本文中我们对鞘脂代谢过程及其代谢产物与冠心病、高血压、心律失常、心力衰竭这4种心血管系统常见病的关系、作用机制进行综述,并对鞘脂通路作为心血管疾病诊治潜在...     

Sox10是一种雪旺细胞和黑色素细胞的广谱标记物

S-100是一种黑色素瘤和外周神经鞘膜细胞肿瘤敏感性标记物。然而，它也在其它一些间叶性和上皮性肿瘤中表达。尽管特异性低，但S-100蛋白在诊断促纤维组织增生性黑色素瘤和外周神经鞘膜肿瘤方面还是很有价值的，因为目前除此之外并无其它特异性标记物可以应用。Sox10是一种神经（管）嵴转录因子，在Schwann细胞和黑色素细胞的分化（specification）、成熟和功能维持方面发挥重要作用。     

CCIO蛋白功能及在疾病中的作用研究进展

Clara细胞是一种分布于哺乳动物肺脏末端细支气管的非纤毛、非浆液分泌上皮细胞,具有多种生物学功能。在气管支气管树分叉处的Clara细胞是支气管上皮细胞的祖细胞,有助于损伤后上皮再生。Clara细胞10．kD蛋白（claraeell10-kDprotein,CCl0）是Clara细胞分泌的一种分子量为10-kD的分泌蛋白。     

透明质酸对大鼠肺泡巨噬细胞和肺成纤维细胞分泌蛋白酶的影响

目的:研究不同大小透明质酸对大鼠肺泡巨噬细胞和肺成纤维细胞分泌基质金属蛋白酶（MMPs）的诱导作用及对细胞周围胶原收缩和降解的影响。方法:Wistar 大鼠肺泡灌洗获取肺泡巨噬细胞,Wistar 乳鼠肺组织块培养获取肺成纤维细胞;透明质酸（简称HA）分为4个分子量：100 kD、200 kD、500 kD、1 000 kD;将两种细胞混悬液分别与胶原溶液、不同分子量的透明质酸溶液混匀形成胶原凝胶后将凝胶漂浮于培养液中连续培养72 h;每24 h 记录凝胶面积;留取第1个24 h的培养上清液应用酶联免疫吸附法检测MMPs;72 h后收集凝胶,应用分光光度测定法进行羟脯氨酸检测。结果:（1）不同分子量HA都可刺激肺泡巨噬细胞分泌MMP-9、MMP-12和MMP-13,与对照组比差异显著(P<0.05);其中以200 kD HA作用最强。（2）不同分子量HA都可刺激肺成纤维细胞分泌MMP-1、MMP-9和MMP-13,与对照组比较,P<0.05;其中以100、200 kD HA作用最为显著。（3）凝胶形成后24 h,100、200、500 kD HA使肺泡巨噬细胞凝胶面积明显缩小,与对照组比较有显著差异（P<0.05),并且这种作用一直持续到72 h。（4）凝胶形成后24 h, 100、200、500 kD的HA使肺成纤维细胞凝胶面积明显缩小,与1 000 kD HA组及对照组比较有显著差异(P<0.05));72 h后,100、200 kD HA使凝胶面积进一步缩小,与500、1 000 kD HA组及对照组比较,P<0.05。（5）4种分子量的HA与肺泡巨噬细胞共同孵育72h后,凝胶中羟脯氨酸含量与对照组比较均有显著减少,其中100、200 kD HA组羟脯氨酸含量降低最显著(P<0.05)。（6）4种分子量的HA与成纤维细胞共同孵育72h后,凝胶中羟脯氨酸含量与对照组比较均显著减少,且不同分子量之间差异显著(P<0.05)。结论：（1）HA可以刺激大鼠肺泡巨噬细胞和肺成纤维细胞分泌多种与胶原降解相关的MMPs;其中200 kD HA作用最强。（2）HA可以促进两种细胞凝胶收缩,以100、200、500 kD的HA作用最显著。（3）HA可以降解细胞凝胶中的胶原,使其羟脯氨酸含量减低,以100、200 kD HA作用最强。     

中华眼镜蛇毒F组份对血小板活化时蛋白酪氨酸磷酸化的影响

目的：观察中华眼镜蛇毒F组份抑制血小板聚集的胞内信号转导机制。方法： 实验分6组：(1)空白组；(2)对照组；(3)-(6)实验组加入浓度为100、30、10、3 mg/L F组份。用比浊法测定血小板聚集率。用蛋白质免疫印迹法（Western blotting）测定血小板内蛋白酪氨酸磷酸化的表达。结果： F组份呈浓度依赖性抑制二磷酸腺苷（ADP）引起的分子量为76、66和37.5 kD蛋白酪氨酸磷酸化的表达，其中30、100 mg/L给药组中，3个分子量蛋白与对照组比较均有显著差异，P<0.05。F组份对ADP引起血小板聚集作用的抑制同降低76、66和37.5 kD 3个分子量蛋白酪氨酸磷酸化的表达呈明显的正相关（r=0.9367,P<0.01）。结论： 中华眼镜蛇毒F组份通过抑制分子量为76、66和37.5 kD蛋白的酪氨酸磷酸化表达，对血小板胞内信号转导过程发生影响，可能是其抗栓机制之一。     

细胞性神经鞘黏液瘤

细胞性神经鞘黏液瘤是一种特殊的组织发生不明确的良性皮肤肿瘤，由于含有不同程度的明显的黏液基质，原来把它归类于真皮的神经鞘黏液瘤（DNSMs）（所谓的黏液样神经鞘黏液瘤）。事实上，尽管没有足够的证据显示其神经鞘分化，但是这些罕见病变却成了病理学家应该诊断什么的问题，甚至有时误诊为恶性肿瘤，由于报道的病例相对较少及有限的随访资料，其临床特点和形态学谱仍未完全明了，其不典型组织学特征的意义尚不清楚。作者分析了1987～2003年间收集的133例细胞性神经鞘黏液瘤的临床病理和免疫组化特征。女性和男性发病比为1．8：1．0，[第一段]     

Parallel Detection and Quantification Using Nine Immunoassays in a Protein Microarray for Drug from Serum Samples

A protein microarray system for detection and quantification of nine prohibited drugs in serum is described. Chemically modified slides were chosen as the microarray substrates because of their suitable for drug-BSA printing. The developed protein microarray was able to preserve the biological function of the haptens, when immobilized on the microarray surface and demonstrated binding with their corresponding antibodies. The microarray could also be used for quantitative analysis when mouse IgG was chosen as an internal control for data processing. There was no qualitative difference between the results obtained using the protein microarray and ELISA. The protein microarray technology should be applicable to performing, simultaneously, large scale screening tests for many different analytes in serum.     

Evidence for an autoprotease activity of sindbis virus capsid protein

Sindbis virus capsid protein is virtually the only product formed when viral 26 S RNA is added to a mouse Krebs ascites cell-free protein synthesis system. However, substitution of arginine and proline by the respective analogues canavanine and azetidine-2-carboxylate inhibits capsid production and larger polypeptides accumulate. The latter are converted to capsid in pulse-chase experiments when the normal amino acids are added during the chase, but not if the chase period contains only the analogues in the reaction mixture. These results support an autoprotease model for the co-translational cleavage of Sindbis virus capsid proteins.     

Stimulation of the cAMP system by the nitric oxide-cGMP system underlying the formation of long-term memory in an insect

The nitric oxide (NO)-cGMP signaling system and cAMP system play critical roles in the formation of multiple-trial induced, protein synthesis-dependent long-term memory (LTM) in many vertebrates and invertebrates. The relationship between the NO-cGMP system and cAMP system, however, remains controversial. In honey bees, the two systems have been suggested to converge on protein kinase A (PKA), based on the finding in vitro that cGMP activates PKA when sub-optimal dose of cAMP is present. In crickets, however, we have suggested that NO-cGMP pathway operates on PKA via activation of adenylyl cyclase and production of cAMP for LTM formation. To resolve this issue, we compared the effect of multiple-trial conditioning against the effect of an externally applied cGMP analog for LTM formation in crickets, in the presence of sub-optimal dose of cAMP analog and in condition in which adenylyl cyclase was inhibited. The obtained results suggest that an externally applied cGMP analog activates PKA when sub-optimal dose of cAMP analog is present, as is suggested in honey bees, but cGMP produced by multiple-trial conditioning cannot activate PKA even when sub-optimal dose of cAMP analog is present, thus indicating that cGMP produced by multiple-trial conditioning is not accessible to PKA. We conclude that the NO-cGMP system stimulates the cAMP system for LTM formation. We propose that LTM is formed by an interplay of two classes of neurons, namely, NO-producing neurons regulating LTM formation and NO-receptive neurons that are more directly involved in the formation of long-term synaptic plasticity underlying LTM formation.     

The Role of Digestive Enzymes in Orally Induced Immune Tolerance

The influence of digestive enzymes on the tolerogenic properties of an orally administered protein antigen, Bovine Serum Albumin (BSA), in the mouse has been investigated. A non-immunogenic peptic digest of BSA was found to be immunosuppressive when administered orally or directly injected into the mouse ileum. In contrast, untreated BSA was tolerogenie when administered orally but immunogenie following ileal administration. As determined by precipitin analysis of the proteins recovered from mouse feces, orally administered BSA was thoroughly degraded by the digestive system while the degradation in the ileum was quite limited. We conclude that to acquire tolerogenic properties, an orally administered protein must be first degraded by the proteolytic enzymes of the gastrointestinal digestive system.     

The role of digestive enzymes in orally induced immune tolerance

The influence of digestive enzymes on the tolerogenic properties of an orally administered protein antigen, Bovine Serum Albumin (BSA), in the mouse has been investigated. A non-immunogenic peptic digest of BSA was found to be immunosuppressive when administered orally or directly injected into the mouse ileum. In contrast, untreated BSA was tolerogenic when administered orally but immunogenic following ileal administration. As determined by precipitin analysis of the proteins recovered from mouse feces, orally administered BSA was thoroughly degraded by the digestive system while the degradation in the ileum was quite limited. We conclude that to acquire tolerogenic properties, an orally administered protein must be first degraded by the proteolytic enzymes of the gastrointestinal digestive system.     

Use of the Sindbis replicon system for expression of LaCrosse virus envelope proteins in mosquito cells

Summary.  The Sindbis replicon expression system was used to express La Crosse (LAC) virus envelope glycoprotein genes in both mammalian and mosquito cell culture. Replicon expressed LAC proteins had correct molecular mass (Mr) and were antigenically similar to wild type LAC envelope proteins. In addition, LAC G1 and G2 proteins colocalized when expressed from separate constructs in both mammalian and mosquito cells suggesting that they were trafficked through the cell similarly to wild type LAC proteins. A truncated form of the G1 protein was secreted from mosquito cells when expressed alone. The truncated G1 protein was also secreted from mosquito cells when expressed with the G2 protein, but to a lesser extent than when expressed alone, suggesting that the G2 protein sequestered G1 protein intracellularly. The Sindbis replicon system is a powerful tool for the study of LAC virus protein maturation within mosquito cells and mosquitoes. Accepted February 2, 1997 Received February 2, 1998     

The Role of Digestive Enzymes in Orally Induced Immune Tolerance

The influence of digestive enzymes on the tolerogenic properties of an orally administered protein antigen, Bovine Serum Albumin (BSA), in the mouse has been investigated. A non-immunogenic peptic digest of BSA was found to be immunosuppressive when administered orally or directly injected into the mouse ileum. In contrast, untreated BSA was tolerogenie when administered orally but immunogenie following ileal administration. As determined by precipitin analysis of the proteins recovered from mouse feces, orally administered BSA was thoroughly degraded by the digestive system while the degradation in the ileum was quite limited. We conclude that to acquire tolerogenic properties, an orally administered protein must be first degraded by the proteolytic enzymes of the gastrointestinal digestive system.     

Transient expression of bacterial gene fragments in eukaryotic cells: implications for CD8(+) T cell epitope analysis

CD8(+) T cells are potent effectors of acquired immunity against some viruses and intracellular bacterial pathogens. Antigens recognized by CD8(+) T cells are small, 8-9 amino acid peptides derived from proteins produced by the pathogen. These peptides are presented by MHC class I molecules on the surface of the infected cell. When characterizing the CD8(+) T cell response to a bacterial or viral pathogen, it is often necessary to express an antigenic protein in a eukaryotic host cell that is capable of processing and presenting peptide epitopes to antigen-specific CD8(+) T cells. We describe a system designed to transiently express bacterial polypeptides and MHC class I molecules in eukaryotic cells. Recognition of these peptide-MHC complexes stimulates TNF production by antigen-specific CD8(+) T cell lines. This system should be useful for analysis of CD8(+) T cell epitope-containing bacterial gene fragments when expression of the entire bacterial protein is detrimental to the eukaryotic cell, or when overexpression of the bacterial gene is detrimental to the bacterial cloning strain. Furthermore, this system can be used for the rapid mapping of CD8(+) T cell epitopes within a protein.     

Highly efficient ribosome display selection by use of purified components for in vitro translation

Ribosome display is a powerful in vitro technology for the selection and directed evolution of proteins. The ribosome display process exploits cell-free translation to achieve coupling of phenotype and genotype by the production of stabilised ribosome complexes in which translated proteins and their encoding mRNA remain attached to the ribosome. Current ribosome display systems that are well proven, by the evolution of high affinity antibodies and the optimisation of defined protein characteristics, use an Escherichia coli cell extract for in vitro translation and display of an mRNA library. Recently, a cell-free translation system has been produced by combining recombinant E. coli protein factors with purified 70S ribosomes. We have applied this development in cell-free translation technology to ribosome display by using the reconstituted system to generate stabilised ribosome complexes for selection. We show that higher cDNA yields are recovered from ribosome display selections when using a reconstituted translation system and the degree of improvement seen is selection specific. These effects are likely to reflect higher mRNA and protein stability and potentially other advantages that may include protein specific improvements in expression. Reconstituted translation systems therefore enable a highly efficient, robust and accessible prokaryotic ribosome display technology.