A novel missense mutation in exon 3 of the COL4A5 gene associated with late-onset Alport syndrome

We have identified a novel missense transition (362G→A) in exon 3 of the COL4A5 gene in a male patient with late-onset Alport syndrome. We used non-isotopic single strand conformation polymorphism, heteroduplex analysis, and automated DNA sequencing. The mutation changes a conserved glycine at codon 54 for an aspartic acid (Gly54Asp), which abolishes a BstNI site. Using restriction analysis, we identified the heterozygous carrier status in the two daughters of the proband. Our findings are in keeping with the hypothesis that slower progressive forms of Alport syndrome are more often associated with missense mutations rather than large deletions or frameshifts. This is the first mutation described in the N-terminus triple helical 7S domain of the COL4A5 gene in an Alport syndrome patient.     

Allelic exclusion of {alpha} chains in TCRs

Although the ß chains of ß⁺ T cells exhibitallelic exclusion it has recently been shown that 20–30%of such cells express both chain alleles on their surface.Potentially these cells have dual specificity, and it has beensuggested that they may play a role in autoimmunity and alloreactlvtty.The analysis presented in this paper examines critically thepossibility that the observed violation of allelic exclusionof chain expression arises as a natural consequence of a chainrearrangement and intrathymlc positive selection. It is concludedthat, subject to certain identified conditions being satisfied,the observed frequency of T cells expressing two chains iscompatible with such a mechanism. However, It is an essentialassumption of the theory that, of the two ß chaincomplexes on these cells, only one mediates positive selection.It follows that to what extent both receptors are actually functionaldepends on whether an ß chain complex that has failedto satisfy the criterion of positive selection can mediate antigenrecognition in the periphery. In principle the answer to thisquestion may be different if one is considering autoreactlvltyor alloreactlvlty. Finally, attention is drawn to the apparentdiscrepancy between the frequency of peripheral T cells expressingtwo chains and the failure to find T cell clones of this phenotype.Possible explanations are discussed.     

Comparative sequence analysis of the M gene among rabies virus strains and its expression by recombinant vaccinia virus

Nucleotide sequences and the deduced amino acid sequences of the gene encoding the matrix (M) protein of the Nishigahara and the CVS strains of rabies virus have been determined. The M gene is 609 nucleotides long and is capable of coding for a peptide composed of 202 amino acids. Sequence comparison of these M genes with those of other stains [Pasteur (PV), ERA, Avol] revealed that there is 89.7–91.5% homology at the nucleotide level, and 90.1–92.1% homology at amino acid level, between almost all combinations of these strains. However, in the combinations of the PV and ERA strains, and the virulent CVS and the avirulent CVS-derived Avol strains, much higher homology was observed both at the nucleotide and amino acid levels. The predicted secondary structure and hydropathy profiles also exhibited similar features. Recombinant vaccinia virus containing the M gene was constructed. Sodium dodecyl sulfate (NaDodSO₄)-polyacrylamide gel electrophoresis of the precipitates obtained by immune reaction of the recombinant virus-infected cell lysate with a monoclonal antibody against the M protein revealed that electrophoretic mobility of the expressed protein is indistinguishable from that of the authentic M protein from rabies virions.The nucleotide sequence data of the M genes of the CVS and Nishigahara (RCEH) strains reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers D90450 and D90451.     

Molecular analysis of structural protein genes of the yamagata-1 strain of defective subacute sclerosing panencephalitis virus. II. Nucleotide sequence of a cDNA corresponding to the P plus M dicistronic mRNA

The nucleotide sequence of a cloned cDNA corresponding to the P+M dicistronic mRNA of a subacute sclerosing panencephalitis (SSPE) virus was determined and compared with data of measles virus (MV). The dicistronic mRNA of the SSPE virus consisted of the 3 proximal 626 nucleotides of P mRNA, intercistronic trinucleotides, a full length of M mRNA, and 75 poly A nucleotides. The part encoding the P protein had a high homology to MV, except at the noncoding region. The terminating consensus sequence of the P gene and the intercistronic trinucleotides of the SSPE virus were CTAC(A)₆ and CCT; in MV they are TTAT(A)₆ and CTT, respectively. In the M gene, the starting consensus sequence was exactly the same as MV, but at the 5 proximal end, one third of this gene was different: The first ATG codon of the MV M gene signaling opening of the reading frame was changed to ACG in the SSPE virus and one long open reading frame started from the third ATG codon. The stop codon (TAG) of the MV M gene was also changed to CAG in the SSPE virus. Thus, the deduced SSPE-virus M protein lacked 50 amino acids at the amino terminal and had 15 extra amino acids at the carboxyl end when compared with the MV M protein.     

Changes in cyclic nucleotides during the calcium paradox in the isolated rat heart

Reperfusion of hearts with a Ca²⁺-containing medium after a perfusion period in Ca²⁺-free medium results in irreversible cell damage (calcium paradox). In this investigation we have studied coronary flow and cyclic AMP and cyclic GMP levels after several periods of Ca²⁺-free perfusion in isolated rat hearts. We also investigated the effects of papaverine (Pap), noradrenaline (NA), acetylcholine (ACh) and absence of inorganic phosphate during Ca²⁺-free perfusion on coronary flow (CF) and cyclic nucleotide levels. Inability of the heart to recover contractile activity with development of contracture during the reperfusion period was accepted as indicative of the calcium paradox. Ca²⁺-free perfusion alone and NA and absence of inorganic phosphate during the Ca²⁺-free perfusion period increased CF, whereas Pap and ACh decreased it. However, only Ca²⁺-free perfusion and NA elevated cyclic AMP. On the other hand, Pap and ACh increased cyclic GMP (with a transient rise of cyclic AMP in Pap infusion), and absence of inorganic phosphate decreased both cyclic AMP and cyclic GMP. Pap, ACh and absence of phosphate prevented the calcium paradox. Our study suggests that increased cyclic AMP during the Ca²⁺-free perfusion may contribute, with the other factors, to the occurrence of the calcium paradox.     

先天性巨结肠患者人类巨细胞病毒UL144基因多态性的研究

目的研究人类巨细胞病毒（human cytomegalovirus,HCMV）UL144基因在先天性巨结肠（Hirschsprung＇s disease,HD）临床株中的多态性,探讨HCMV UL144基因多态性与致病性之间的关系.方法随机选取53个先天性巨结肠患儿痉挛段结肠手术标本及经荧光定量PCR方法检测HCMV DNA为阳性的4个HD患儿的尿标本,对照组为无症状或仅有皮肤轻度黄疸的6个尿标本.应用巢式聚合酶链反应的方法,扩增HCMV UL144基因开放阅读框架（ORF）,扩增阳性的临床株进行双向DNA测序,最后通过DNAclub、Bioedit、DNAstar、GeneDoc等软件进行分析.结果23份HD痉挛段肠组织（46%）及4份尿标本HCMV UL144基因扩增阳性,并且完成测序.种系进化树分析结果显示25个HD患儿的DNA序列分为3个基因型,G1A型64.0%,G2型24%,G3型12%.与对照组比较,经χ^2检验,χ^2=10.93,P为0.012;其中HD临床株G1A和G3型基因经Fisher检验,P为0.015,差异具有统计学意义.全结肠型、长段型及普通型HD分散分布于UL144各个基因型中.结论HD与HCMV感染有关,HCMV可能是HD的病因之一;在HD患儿中,HCMV感染以UL144基因G1A型为主;HD的临床分型与HCMV UL144基因分型无关.     

小发卡RNA抗呼吸道合胞病毒效应的研究

目的 为从基因水平抑制呼吸道合胞病毒（respiratory syncytial virus，RSV）的复制，针对RSV的M2-2基因构建小发卡结构状RNA（short hairpin RNA，shRNA）重组质粒，在细胞水平观察shRNA对RSV复制的影响。方法 将成功构建的针对RSV M2-2基因的重组质粒pshRNA8260转染HEp-2细胞，利用光镜观察pshRNA8260对RSV致HEp-2细胞病变效应（cytopathogenic effect，CPE）的影响并计算CPE抑制率，空斑形成实验检测RSV滴度变化。结果 成功构建了针对人RSV M2-2基因mRNA的pshRNA8260重组质粒，研究发现pshRNA8260能明显改善RSV所致的病变效应，降低RSV在细胞内复制的病毒滴度。结论 针对RSV M2-2基因的pshRNA82（g）重组质粒具有明显的特异性抗RSV效应的作用。     

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.     

beta2-adrenergic receptor gene polymorphisms in myasthenia gravis (MG)

The beta2-adrenergic receptor (beta2-AR) belongs to the group of G-protein coupled receptors and is present mainly on skeletal and cardiac muscle cells and lymphocytes. The gene encoding beta2-AR (ADRB2) displays a moderate degree of heterogeneity in the human population. The distribution of polymorphisms at amino acid positions 16, 27 and 164 is changed in asthma, hypertension and obesity. We have earlier reported a decreased density of the beta2-AR on peripheral blood mononuclear cells and the presence of beta2-AR antibodies in patients with MG. Since certain polymorphisms affect the function of the beta2-AR, it was of interest to analyse these in MG. Using allele-specific polymerase chain reaction amplification, we revealed an over-representation of homozygosity for Arg16 and a lower prevalence of homozygosity for Gly16 in MG patients compared with healthy individuals. The increased frequency of homozygosity for Arg16 was due to a contribution from patients with generalized MG but not from patients with only ocular disease. Homozygosity for Glu27 was negatively associated with both the presence of beta2-AR antibodies and severity of disease. Moreover, acetylcholine receptor (AChR) antibodies were more often present in patients being homozygous for Gln27. Our results imply that homozygosity for Arg16 confers susceptibility to generalized MG, and that certain polymorphisms at amino acid position 27 are associated with subgroups of patients.