Genetics of reovirus: the relationship of interference to complementation and reassortment of temperature-sensitive mutants at nonpermissive temperature.

Temperature-sensitive mutants of reovirus type 3 are capable of interfering with the replication of wild-type reovirus type 3. The interfering activity correlated with the ability of pairs of mutants to complement at 39°: Pairs of noninterfering mutants (tsD × tsE) yielded efficient complementation (indexes of 10–50); pairs of interfering mutants (including members of groups ts A, B, G) did not produce significant complementation (indexes ～ 1). The ability of pairs of mutants to reassort at 39° generally followed a similar pattern. Thus interference is an important property of ts mutants of reovirus and needs to be considered when genetic interactions are being studied at 39°.     

Soluble and membrane-bound forms of brain acetylcholinesterase in Alzheimer''s disease

In order to determine the effect of Alzheimer's disease on the relative distribution of soluble and membrane-bound molecular forms of acetylcholinesterase (AChE) in the brain, postmortem samples (delay interval less than 12 h) were obtained from parietal cortex (Brodmann area 40) and hippocampus as well as the areas containing their respective projection nuclei, i.e., substantia innominata and septal nucleus, in 9 patients with Alzheimer's disease (AD) and 4 normal controls. The monomer (G₁), dimer (G₂), and tetramer (G₄) forms of AChE were examined. In AD compared to controls, significant changes occurred in area 40 and hippocampus but not in the areas containing projection nuclei, and included loss of mean total AChE activity, decrease in the relative percentage of membrane-bound G₄, and increase in the relative percentage of soluble G₁---G₂. Percent of soluble G₄ was unaffected in AD brain. In area 40 but not hippocampus a large increase in percent membrane-bound G₁-G₂ occurred. Thus, these results emphasize that the selective decrease in membrane-bound G₄ accounts for the decrease in total G₄ activity in AD brain.     

Quantitative changes in gene expression of glutamate receptor subunits/subtypes in the vestibular nucleus, inferior olive and flocculus before and following unilateral labyrinthectomy in the rat: real-time quantitative PCR method

Spontaneous recovery from the oculomotor and postural symptoms of unilateral labyrinthectomy (UL) is known as vestibular compensation, which is a useful model for investigation of the mechanisms of lesion-induced CNS plasticity. In the present study, to elucidate the molecular biological basis of vestibular compensation, we investigated changes in the mRNA expression of glutamate receptor subunit/subtypes in the rat central vestibular system, including the vestibular nucleus complex (VNC), inferior olive (IO), and cerebellar flocculus following UL, using a real-time quantitative polymerase chain reaction (PCR) method. In normal control animals, regional differences in the expression of several glutamate receptor subunit/subtypes, e.g., NR1 and NR2A subunits of the N-methyl-D-aspartic acid (NMDA) receptor, GluR2 and KA2 subtypes of non-NMDA receptors, and mGluR1 and mGluR7 metabotropic glutamate receptors, were consistent with previous results from studies using in situ hybridization histochemistry, suggesting that the real-time quantitative PCR method was a reliable procedure for evaluation of changes in mRNA expression. In the vestibular nucleus complex, NR2A, GluR2 and mGluR7 mRNA were ipsilaterally downregulated by 6 h following UL (P<0.05, P<0.05 and P<0.01, respectively). In the inferior olive, no changes in gene expression were observed. In the ipsilateral flocculus, KA2 mRNA expression was increased by 50 h post-UL (P<0.05). However, in the contralateral flocculus, mGluR1 mRNA was downregulated by 6 h post-UL (P<0.005). Both the increase in KA2 mRNA expression in the ipsilateral flocculus and the decrease in mGluR1 mRNA expression in the contralateral flocculus may have had the effect of reducing Purkinje cell inhibition of ipsilateral VNC neurons, thereby contributing to the rebalancing of spontaneous resting activity between the ipsilateral and contralateral VNCs. It is suggested that such changes in the activities of the floccular-VNC pathways may be important to the vestibular compensation process. Electronic Publication     

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

Three novel missense mutations in the human lysosomal sialidase gene causing amino acid substitutions (P80L, W240R, and P316S) in the coding region were identified in two Japanese sialidosis patients. One patient with a severe, congenital form of type 2 sialidosis was a compound heterozygote for 239C-to-T (P80L) and 718T-to-C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitution of 946C-to-T (P316S). None of these mutant cDNA products showed enzymatic activity toward an artificial substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distribution when coexpressed with the PPCA gene in COS-1 cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded in the lysosomes. Homology modeling of the structural changes introduced by the mutations predicted that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The W240R substitution was deduced to influence the molecular surface structure of a limited region of the constructed models, which was also influenced by previously identified V217M and G243R transversions. Received: Stptember 21, 2001 / Accepted: November 2, 2001     

Novel mutations, including the second most common in Japan, in the β-hexosaminidase α subunit gene, and a simple screening of Japanese patients with Tay-Sachs disease

Two novel mutations of the β-hexosaminidase α subunit gene were identified in Japanese patients with the infantile form of Tay-Sachs disease. One mutation was a one-base deletion at nt613C, which generated a stop codon at two codons downstream, in three unrelated patients. The other mutation was a one-base substitution of G-to-A at IVS 5, +1, which caused a splicing abnormality, in one patient. A missense mutation of R170W, which has already been reported in other ethnic groups, was also newly identified in one patient. In 1993, the most common mutation (IVS 5, −1G → T) in Japanese patients with Tay-Sachs disease was reported as the major mutation in Japan accounting for 80% of 56 mutant alleles from 28 unrelated patients. The deletion of nt613C was the second most common mutation, accounting for 5% of the mutant alleles. The previously reported mutation IVS 5, −1G → T and the nt613C deletion found in this study together accounted for 85% of the mutations causing Tay-Sachs disease among Japanese. Since these two mutations were located in or close to exon 6 and since they abolish Fok I (IVS 5, −1G → T) and Sfa NI (nt613C deletion) restriction sites, respectively, they were screened rapidly by single polymerase chain reaction followed by digestion with these enzymes. Received: November 10, 1998 / Accepted November 14, 1998     

Molecular typing of echovirus serotype 4 isolates

We report on clinical samples Stuttgart/97, Berlin/99 and Jasi/99 associated with aseptic meningitis. All three samples contained echovirus 4 (E4) but Stuttgart/97 was simultaneous infected with echovirus 30 (E30). The genetic relationship of the E4 strains was assessed using RT-PCR and direct sequencing of amplicons derived from the genomic region encoding the capsid protein VP1. The sequences have been compared with each other and with sequences of further E4 strains obtained from GenBank. The analysis confirms that sequences of recent isolates have drifted away from elderly strains over a longer period of time. Several amino acid changes in assumed antigenic sites of the VP1 gene may be sufficient to cause changes in antigenic specificity and therefore they may be a reason for failure of serological typing of some new antigenic E4 variants.     

常见呼吸道病毒分子鉴别诊断技术的建立

目的 建立一种常见呼吸道感染病毒的快速检测方法,为尽早诊断、减少疾病的传播以及为临床提供良好的治疗依据.方法 采用液体芯片检测技术结合靶向多重RT-PCR技术建立可以同时检测13种呼吸道病原的检测技术.结果 该方法特异性方面检测13种常见呼吸道病毒没有交叉,标本的检出率为100%;灵敏度方面达到10e2-10e1（pfu/ml）.结论 该分子鉴别诊断可应用常见呼吸道病毒的检测,协助诊断病毒引起的病毒性呼吸道感染.     

Genome analysis of dengue type-1 virus isolated between 1990 and 2001 in Brazil reveals a remarkable conservation of the structural proteins but amino acid differences in the non-structural proteins

We have investigated the genetic diversity of dengue type-1 (DEN-1) virus in Brazil. The full nucleotide sequences of three DEN-1 virus isolated from DEN fever (DF) and DEN hemorrhagic fever patients in northeastern Brazil in 1997 (BR/97) and one from a DF patient in the south of Brazil in 2001 (BR/01) were compared to that of the reference strain BR/90 obtained in the city of Rio de Janeiro in 1990. Sequence analysis showed that the structural proteins were remarkably conserved between all isolates. A total of 27 amino acid changes occurred throughout the non-structural proteins. Among them, nine amino acid substitutions were specific of BR/97 and BR/01 isolates, indicating that in situ evolution of these strains had occurred. Within the BR/97 and BR/01 samples, some amino acid substitutions have been previously identified in DEN-1 virus strains sequenced so far, suggesting that recombination events might have occurred.     

2001至2005年上海部分地区腹泻住院患儿诺若病毒分子临床流行病学研究

目的通过对上海地区腹泻住院患儿进行诺若病毒检测,对其流行株进行基因序列的测定,以了解诺若病毒在上海地区的流行特征,为该病原体所致腹泻的防治提供基础数据和理论依据。方法收集2001至2005年复旦大学附属儿科医院5岁以下腹泻住院患儿的粪便标本。首先进行轮状病毒的检测,在轮状病毒抗原阴性标本中,每隔8个标本按编号顺序行机械随机抽样,建立RT-PCR方法进行诺若病毒的检测。对PCR产物进行双向测序,测序结果通过Clustal W和Mega 4.1软件进行分析。结果研究期间共收集腹泻患儿粪便标本5534份,轮状病毒抗原阴性4084份,机械随机抽得484份用于诺若病毒检测,45/484份(9.3%)检测到诺若病毒。对诺若病毒感染季节分布和患儿年龄特点的分析表明,除4月和7月份未检测到诺若病毒外,其余各月份均检测到诺若病毒,其高发的月份是8至11月。5~6月也呈一个小高峰。77.8%(35/45)的患儿<2岁,其中6~11个月的患儿所占比例最高,达35.6%(16/45),<6个月的婴儿占20%(9/45)。GⅡ-4型是这5年间尤其是2003年之后的主要流行型别,2001至2002年尚存在其他的流行型别GⅡ-3和GⅡ-7...     

阳离子交换色谱柱上聚合血红蛋白研究

采用柱上聚合的方法制备血红蛋白溶液，解决传统血红蛋白聚合过程中由于戊二醛的活性过高导致平均分子量大、产物分子量分布宽的问题。该方法利用阳离子交换剂对修饰度（聚合度）小的血红蛋白吸附能力大的原理，使其在柱上富积，同时加入戊二醛进行聚合反应。结果表明该方法能比较有效地缩小聚合血红蛋白的分子量分布。