Polycystin-1, the PKD1 gene product, is in a complex containing E-cadherin and the catenins

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by cyst formation in kidney tubules and other ductular epithelia. Cells lining the cysts have abnormalities in cell proliferation and cell polarity. The majority of ADPKD cases are caused by mutations in the PKD1 gene, which codes for polycystin-1, a large integral membrane protein of unknown function that is expressed on the plasma membrane of renal tubular epithelial cells in fetal kidneys. Because signaling from cell-cell and cell-matrix adhesion complexes regulates cell proliferation and polarity, we speculated that polycystin-1 might interact with these complexes. We show here that polycystin-1 colocalized with the cell adhesion molecules E-cadherin and alpha-, beta-, and gamma-catenin. Polycystin-1 coprecipitated with these proteins and comigrated with them on sucrose density gradients, but it did not colocalize, coprecipitate, or comigrate with focal adhesion kinase, a component of the focal adhesion. We conclude that polycystin-1 is in a complex containing E-cadherin and alpha-, beta-, and gamma-catenin. These observations raise the question of whether the defects in cell proliferation and cell polarity observed in ADPKD are mediated by E-cadherin or the catenins.     

外源重组基因表达产物亚细胞定位的研究现状

目的：将外源基因通过重组然后在宿主细胞中表达,以研究其功能.资料来源：应用计算机检索Medlinel997-01/2004-12与外源基因重组表达产物亚细胞定位的相关文献,检索词“foreign recombined gene,subcellure location”等分别进行检索提炼,限定文献语种为English.资料选择：就检索到的500余篇资料进行初审,纳入标准：①有关外源基因重组策略.②与表达产物定位检测方法相关.排除标准：文献中重复研究、综述、Meta分析类文章.未排除文章中资料是否应用了随机、对照和盲法.资料提炼：共收集到80余篇关于外源基因重组表达后定位相关的文章.其中研究内容相似的,以近5年内发表在较权威杂志者优先.对符合标准的38篇文献进行分析.资料综合：外源重组基因在宿主细胞中表达后,可以通过多种方法进行亚细胞定位,如报告基因,免疫电镜,免疫荧光技术等,且大规模的外源重组基因亚细胞定位具有重要的意义.结论：因为外源重组基因表达产物的功能与其在宿主细胞中的定位有重要的关系,所以研究其在宿主细胞中的亚细胞定位,对研究外源重组基因表达产物的功能或未知新基因的功能来说有很重要的意义.     

外源重组基因表达产物亚细胞定位的研究现状

目的：将外源基因通过重组然后在宿主细胞中表达，以研究其功能。 资料来源：应用计算机检索Medline 1997-01／2004-12与外源基因重组表达产物亚细胞定位的相关文献，检索词“foreign recombined gene，subcellure location”等分别进行检索提炼，限定文献语种为English。 资料选择：就检索到的500余篇资料进行初审，纳入标准：①有关外源基因重组策略。②与表达产物定位检测方法相关。排除标准：文献中重复研究、综述、Meta分析类文章。未排除文章中资料是否应用了随机、对照和盲法。 资料提炼：共收集到80余篇关于外源基因重组表达后定位相关的文章。其中研究内容相似的，以近5年内发表在较权威杂志者优先。对符合标准的38篇文献进行分析。 资料综合：外源重组基因在宿主细胞中表达后，可以通过多种方法进行亚细胞定位，如报告基因，免疫电镜，免疫荧光技术等，且大规模的外源重组基因亚细胞定位具有重要的意义。 结论：因为外源重组基因表达产物的功能与其在宿主细胞中的定位有重要的关系，所以研究其在宿主细胞中的亚细胞定位，对研究外源重组基因表达产物的功能或未知新基因的功能来说有很重要的意义。     

Identification of new polymorphisms in the CACNA1S gene.

We identified four novel polymorphisms in the CACNA1S gene that encodes the alpha1-subunit of the dihydropyridine receptor. Mutations in this gene are associated with two genetic diseases: malignant hyperthermia and hypokalemic periodic paralysis. The nucleotide substitutions c2403T --> C and c5398T --> C did not result in amino acid replacement, the nucleotide substitution c4475C --> A caused the replacement of the Ala1492 with an Asp residue and an A insertion was identified in intron 36. By using methods based on digestion with restriction enzymes we calculated the frequencies of these novel polymorphisms, as well as heterozygosity, in normal subjects from southern Italy.     

A common polymorphism in exon 46 of the human autosomal dominant polycystic kidney disease 1 gene (PKD1)

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common single gene diseases in humans. We have identified a synonymous T to C transition polymorphism in exon 46 of the PKD1 gene (12838T→C, Pro4209Pro). The polymorphism was present with similar frequencies in ADPKD patients and unaffected individuals. The heterozygosity, determined in 89 Italian individuals, was 0·347. The frequency of the rarer allele was 0·222. This polymorphism is easy to determine as it abolishes a naturally occurring DdeI restriction site. The availability of an additional intragenic marker in the PKD1 gene will improve the accuracy of linkage studies in ADPKD families.     

Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters.

The expression of leptin, the ob gene product, is increased in adipose tissue in response to feeding and energy repletion, while leptin decreases food intake. Because adipose tissue gene expression is regulated by cytokines induced during infection and because infection is associated with anorexia, we tested whether induction of leptin might occur during the host response to infection. Administration of endotoxin (LPS), a model for gram negative infections, induces profound anorexia and weight loss in hamsters. In fasted adipose tissue to levels similar to fed control animals. There is a strong inverse correlation between mRNA levels of leptin and subsequent food intake. TNF and IL-1, mediators of the host response to LPS, also induced anorexia and increased levels of leptin in mRNA in adipose tissue. As assessed by immuknoprecipitation and Western blotting, circulating leptin protein is regulated by LPS and cytokines in parallel to regulation of adipose tissue leptin mRNA. Induction of leptin during the host response to infection may contribute to the anorexia of infection.     

Polymorphisms in the human autosomal dominant polycystic kidney disease 2 (PKD2) gene.

Three polymorphisms of the PKD2 (MIM 173910) gene in patients with autosomal dominant polycystic kidney disease are reported: (1) a substitution from ATT (isoleucine) to GTT (valine) at codon 452; (2) a substitution from CGG (arginine) to CAG (glutamine) at codon 848; and (3) a substitution from G to A in intron 4 of the gene. The minor allelic frequencies of codon 452 and intron 4 in the Korean population were estimated to be 0.03 and 0.32, respectively. Although the codon 848 substitution was not observed in 45 unrelated healthy Korean people, the substitution did not cosegregate with the disease phenotype, suggesting that this was a rare, non-deleterious alteration.     

Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.     

原癌基因LMO2短剪切模式的结合蛋白鉴定及功能分析

目的 研究原癌基因LMO2短剪切模式(LMO2-C)的结合蛋白在白血病细胞K562中的表达及功能分析.方法 利用麦芽糖结合蛋白(MBP)沉淀技术和哺乳动物双杂交技术研究LMO2-C的结合蛋白在K562细胞中的表达;半定量RT-PCR检测过表达LMO2-C的K562细胞中红系发育的标志性基因GPA的表达水平.结果MBP原核表达系统成功表达并被纯化出可溶性MBP-LMO2-C结合蛋白,利用MBP沉淀技术验证了LMO2-C能与K562细胞中内源性的GATA-1、LDB1结合;哺乳动物双杂交试验进一步证明LMO2-C能与LDB1结合,而且其过表达能够抑制LMO2-L与LDB1的结合,抑制率达(81.13±0.68)%;半定量RT-PCR结果显示在过表达LMO2-C的K562细胞中,GPA基因相对表达水平下调(51.00±1.58)%.结论LMO2-C能结合K562细胞中内源性GATA-1、LDB1蛋白,并能下调红系发育的标志性基因GPA的表达.     

汉族人多囊肾病1型致病基因突变检测体系的建立及初步应用

目的 建立适合筛查汉族人多囊肾病1型致病基因（PKD1）突变的检测体系。方法 利用设计的82对引物[8对针对PKD15′端多拷贝区的长链聚合酶链式反应（PCR）引物和57对巢式PCR引物，17对针对3′端单拷贝区PCR引物]分别对PKD1的46个外显子进行扩增，扩增产物通过单链构象多态性（SSCP）分析筛检出异常条带后，再经测序确定基因突变位点。利用建立的PCR－SSCP检测体系对汉族人2个常染色体显性遗传性多囊肾病患者家系进行PKDA1突变检测，健康献血员为对照。结果 用82对PCR引物，可成功扩增PKD1各个外显子区域，并经测序证实为PKD1目的片段。将建立的SSCP－PCR基因突变检测体系，分别从2个汉族人常染色体显性多囊肾病（ADPKD）家系检测出PKD1基因Del 3 bp(G49761-G49763)和C47629T2个突变，其可分别导致编码产物第3827位缺失谷氨酸（Glu3827)和第3555位丝氨酸，而产生由苯丙氨酸(S3555F)替代的改变。结论 本研究建立的PCR－SSCP检测体系，可完成PKD1各外显子区域特异性扩增，并成功检测出汉族人2个ADPKD家系基因突变位点，不仅为PKD1基因突变的致病机理研究提供宝贵资料，而且为下一步汉族人多囊肾病的大规模基因突变筛查和临床诊断试剂盒的研制奠定了基础。     

靶向序列捕获联合高通量测序鉴定成人型多囊肾PKD1基因新突变

目的 探讨一个成人型多囊肾蛋白激酶D1(PKD1)基因新剪切位点突变及临床表型.方法 收集一个成人型多囊肾病家系临床资料,对家系内成员采用靶向序列捕获对PKD1基因行高通量测序,并结合文献加以分析.结果 该家系中先证者存在成人型多囊肾PKD1基因的c.12138+1G>A(IVS44)突变,而在家系内父亲因多囊肾引发尿...     

Characterization of a staphylococcal trimethoprim resistance gene and its product.

Resistance to trimethoprim (Tp) is mediated by a plasmid-encoded gene in staphylococci. The gene is responsible for high-level resistance (MIC, greater than 1,000 micrograms/ml) in both its native host and when cloned on high-copy-number vectors in Escherichia coli. Analysis of subclones of the staphylococcal Tp gene on E. coli expression vectors and estimation of the size of full and truncated proteins produced in E. coli minicells generated an approximate size limit of 505 base pairs for the gene and 18,500 daltons for the gene product. Crude extracts of E. coli containing the cloned gene had dihydrofolate reductase (DHFR) specific activity that was more than 100 times greater than that of control cells and more than 1,000 times more resistant to trimethoprim inhibition. The amount of trimethoprim required for a 50% reduction in the specific activity of staphylococcal DHFR differed from those of cells containing DHFR types I, II, or III, enzymes mediating Tp in members of the family Enterobacteriaceae. In addition, the size of the monomeric staphylococcal DHFR protein was larger than that of any of the gram-negative DHFRs both compared with published sequence data and as observed by direct comparison on polyacrylamide gels. Finally, there was no homology between a DNA fragment containing the cloned staphylococcal gene and DNA encoding any of the gram-negative DHFRs. Thus, the staphylococcal Tp gene codes for a single protein with DHFR activity that appears to be unrelated to DHFR genes that mediate Tp in members of the Enterobacteriaceae.     

Identification of a gC1q-binding protein (gC1q-R) on the surface of human neutrophils. Subcellular localization and binding properties in comparison with the cC1q-R.

Human neutrophils have multiple C1q-binding proteins. Direct ligand-binding studies with the globular domain of C1q and two-dimensional Western blot analysis revealed two gC1q-binding proteins (gC1q-R): a 33,000 M(r) protein (pI 4.5) mainly in the neutrophil plasma membrane and an 80,000-90,000 M(r) protein (pI 4.1-4.2) located mainly in the granules. Direct binding studies showed that C1q bound to this higher molecular weight protein under physiological conditions. In contrast, anti-cC1q-R antibody, which recognizes a protein binding to collagenous tails of C1q, detected only a 68,000 M(r) protein in the plasma membrane. Both the 33,000 and 68,000 M(r) receptors appear early on the surface of differentiating HL-60 cells. On mature neutrophils, surface expression of both C1q receptors was evident, but no upregulation was observed upon stimulation. Phorbol myristate acetate treatment of neutrophils downregulated both the receptors from cell surface, and significant amounts of soluble gC1q-R were in cell media supernatants, suggesting receptor shedding or secretion. gC1q-R, unlike cC1q-R, did not bind to other C1q-like ligands, namely mannose binding protein, surfactant protein-A, surfactant protein-D, or conglutinin under normal ionic conditions, suggesting a greater specificity for C1q than the "collectin" type receptor (cC1q-R). Rather, gC1q-R only bound purified C1q, and the binding was enhanced under low ionic conditions and in the absence of calcium. The role of C1q receptor shedding and its biologic consequence remain to be defined, but may contribute to the diversity of C1q-mediated responses observed in many cell types.     

Identification and subcellular localization of human rab5b, a new member of the ras-related superfamily of GTPases.

Members of the mammalian rab family of GTPases are associated with specific subcellular compartments, where these proteins are postulated to function in vesicular transport. By screening a human umbilical vein endothelial cell library with degenerate oligonucleotide probes, we have isolated a 1.6-kb cDNA clone encoding a 215-amino-acid protein belonging to the rab family of GTPases. This newly identified rab protein is 81% identical to human rab5, the canine counterpart of which has been localized to the plasma membrane and early endosomes. In light of this homology, we have named this new member of the GTPase superfamily "rab5b." Northern analysis using the rab5b cDNA as a probe revealed a 3.6-kb mRNA in a variety of cell types, including human umbilical vein endothelial cells, K562 erythroleukemia cells, U937 monoblastic cells, and HeLa cells. A fusion protein between glutathione-S-transferase (GST) and rab5b was expressed in bacteria and purified to homogeneity. The recombinant protein was shown to bind GTP and GDP. As is typical of other recombinant rab proteins, the rab5b-GST fusion protein displayed a low intrinsic rate of GTP hydrolysis (0.005/min). An antiserum to rab5b was prepared and used to determine the apparent molecular size and subcellular distribution of the protein. Western blotting with this antibody revealed a 25-kD protein in COS cells transfected with rab5b and in nontransfected HeLa cells. Indirect immunofluorescence and subcellular fractionation showed that rab5b localizes to the plasma membrane. We speculate that rab5b plays a role in vesicular trafficking at the plasma membrane in various cell types.     

Chromosomal location of the genes encoding the leukocyte adhesion receptors LFA-1, Mac-1 and p150,95. Identification of a gene cluster involved in cell adhesion

The adhesion receptors Mac-1, LFA-1, and p150,95 are cell surface alpha/beta heterodimers that play a key role in leukocyte adhesion processes. The genes for Mac-1, LFA-1, and p150,95 alpha subunits have been located to chromosome 16 by means of Southern blot analysis using a series of somatic cell hybrids. Chromosomal in situ hybridization has demonstrated that the genes for the three alpha subunits map to the short arm of chromosome 16, between bands p11 and p13.1, defining a cluster of genes involved in leukocyte adhesion. The gene encoding the LFA-1/Mac-1/p150,95 beta subunit, and defective in leukocyte adhesion deficiency, has been located on chromosome 21, band q22. The leukocyte adhesion receptor alpha and beta subunits are mapped to chromosomal regions that have been shown to be involved in cytogenetic rearrangements in certain patients with acute myelomonocytic leukemia and the blast phase of chronic myelogenous leukemia, respectively.