Production of anti-NC1 antibody by affected male dogs with X-linked hereditary nephritis: A probe for assessing the NC1 domain of collagen type IV in dogs and humans with hereditary nephritis

Summary Some patients with hereditary nephritis (HN) who have received a renal transplant have been shown to form antibody with specificity for the NC1 domain of collagen type IV, a major constituent of glomerular basement membranes (GBM). We attempted to duplicate this phenomenon in a family of dogs with X-linked HN, a model for human X-linked HN, by immunizing affected male dogs with normal dog NC1 domain. A collagenase digest was prepared from normal dog GBM, the NC1 domain was separated into dimer (50 kDa) and monomer (24 kDa and 26 kDa) components by SDS-PAGE, and injected into two affected male dogs. Antisera obtained from both dogs contained antibody which reacted with the NC1 domain of dog and human GBM by a plate-binding radioimmunoassay, bound to the dimer and 26 kDa monomer bands by Western blotting, and staining dog and human GBM by immunofluorescence (IF). The affected male dog antiserum reacted equally by radioimmunoassay with the NC1 domain isolated from GBM of unaffected, affected male, and carrier female dogs in the family with X-linked HN, and bound by Western blotting to dimers and the 26 kDa monomer band of the NC1 domain of GBM in each group of dogs. However, the affected male dog antiserum differentiated these dogs by IF; it produced global staining of GBM of unaffected dogs, failed to stain GBM of affected male dogs, and produced segmental staining of GBM of carrier female dogs. Absorption of the affected male dog antiserum with normal dog NC1 domain eliminated the staining of dog GBM by IF, whereas staining persisted after absorption with affected male dog NC1 domain. The abnormal staining patterns of GBM seen by IF in the affected male and carrier female dogs and the results of the absorption studies imply an abnormality of one or more determinants in the 26 kDa monomer band of the NC1 domain of their GBM. Amino acid sequencing of this band identified the 1(IV) chain of collagen type IV, a finding that has implications for the pathogenesis of canine X-linked HN. Absent and segmental staining respectively were also seen by IF in GBM of a male and female patient with HN, using the affected male dog antiserum. Thus, the results obtained in affected male and carrier female dogs with X-linked HN may also be relevant to patients with this disease.     

A novel missense mutation (I344K) in the SPG4gene in a Korean family with autosomal-dominant hereditary spastic paraplegia

 Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower extremities. Among eight loci linked with autosomal-dominant (AD)-HSP, the SPG4 locus on chromosome 2p22 accounts for about 40% of all patients. Recently, mutations in a new member of the AAA protein family, called spastin, have been identified as responsible for SPG4-linked AD-HSP. Here, we describe a novel missense mutation (c.1031T>A; I344K) in exon 7 of the SPG4 gene identified in a Korean family with typical clinical features of pure AD-HSP. The mutation affects the third amino acid of the highly conserved AAA cassette domain, which is the most fore part of the domain altered by a missense mutation reported so far. Clinical presentations of affected individuals carrying the I344K mutation were not different from those of pure AD-HSP with SPG4 mutations reported previously. However, it is noteworthy that neither urinary dysfunction nor involvement of upper extremities was noticed in this family. To our knowledge, this is the first report of genetically confirmed AD-HSP in Korea. Received: February 20, 2002 / Accepted: May 21, 2002     

Molecular identification of a novel human rotavirus in relation to subgroup and electropherotype of genomic RNA

A total of 41 stool rotavirus specimens collected from children with acute diarrhea at four different locations in Akita Prefecture, Japan, during the peak of the winter diarrhea epidemic in 1988 were analyzed by polyacrylamide gel electrophoresis of viral RNA in conjunction with subgrouping assay. We found that a single strain predominated, with cocirculating strains with less common electropherotypes at a given location, and that two different strains could predominate at geographically close but different locations even during a very limited time of the epidemic season. Furthermore, we isolated a human rotavirus strain (AU125) that was similar to the AU-1 strain in that it possessed a long RNA pattern yet belonged to subgroup I. Genetic analysis by RNA-RNA hybridization assay indicated that the AU125 strain was distinct from two previously identified human rotavirus gene groups (genogroups) represented by the Wa strain (subgroup II with long RNA electropherotype) and the DS-1 strain (subgroup I with short RNA electropherotype), but was very closely related to the AU-1 strain. These data suggest that the genetic diversity of human rotaviruses may be more extensive than was previously thought.     

Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.     

Argonaute蛋白家族在RNAi中的作用

RNA干扰是一种双链RNA诱导的基因沉默现象。Argonaute蛋白组成了一个高度保守的家族,该蛋白家族包括许多成员,它们组成了RNA诱导的沉默复合体的核心元件,是RNA干扰所必须的。Argo-naute蛋白选择性地与miRNA和siRNA结合,并与Dicer酶相互作用,其PIWI盒子与Dicer的RNaseⅢ结构域直接相互作用,PIWI与Dicer之间的相互作用可能会促进miRNA/siRNA的释放。Argonaute蛋白很可能是RNA干扰中核酸内切酶活性的执行者。     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

大鼠重组IgE Fc区CH2-3的原核表达及活性研究

目的 研究大鼠IgE的Fc区CH2-3的生物学活性。方法 构建大鼠IgE的Fc区CH2-3的原核表达质粒pBAD／gⅢA／Ch2-3，并转化入TOP10中，阿拉伯糖诱导表达、周质腔抽提、Ni-NTA金属鳌合柱纯化获得大鼠IgE的Fc区CH2-3，细胞及动物水平检测蛋白质生物学活性。结果 原核系统表达出大鼠IgE的Fc区CH2-3，它能够阻断OVA激发的RBL-2H3的脱颗粒反应，阻断被动皮肤实验。结论 大鼠IgE的Fc区CH2-3能够封闭IgE高亲和力受体，阻断过敏反应。     

Nonlinear and Frequency-Dependent Mechanical Behavior of the Mouse Respiratory System

The assessment of the mechanical properties of the respiratory system is typically done by oscillating flow into the lungs via the trachea, measuring the resulting pressure generated at the trachea, and relating the two signals to each other in terms of some suitable mathematical model. If the perturbing flow signal is broadband and not too large in amplitude, linear behavior is usually assumed and the input impedance calculated. Alternatively, some researchers have used flow signals that are narrow band but large in amplitude, and invoked nonlinear lumped-parameter models to account for the relationship between flow and pressure. There has been little attempt, however, to deal with respiratory data that are both broadband and reflective of system nonlinearities. In the present study, we collected such data from mice. To interpret these data, we first developed a time-domain approximation to a widely used model of respiratory input impedance. We then extended this model to include nonlinear resistive and elastic terms. We found that the nonlinear elastic term fit the data better than the linear model or the nonlinear resistance model when amplitudes were large. This model may be useful for detecting overinflation of the lung during mechanical ventilation. © 2003 Biomedical Engineering Society. PAC2003: 8719Rr, 8719Uv