Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.     

Identification of mutations preventing n-hexadecane uptake among 26 n-alkane non-utilizing mutants of Yarrowia (Saccharomycopsis) lipolytica

Summary Genetic analyses of n-alkane non-utilizing mutants of the yeast Yarrowia (Saccharomycopsis) lipolytica were continued. By analyses of inter-mutant complementation and recombination a total of 26 genetic loci have been identified. Mutations representing these loci have phenotypes characteristic of defects in substrate uptake or in one or more of the enzymatic activities making up the hydroxylase complex. Tests of ¹⁴C n-hexadecane uptake by a set of alkane-negative mutants representing the 26 loci show that 16 of the mutations cause a significant reduction in n-alkane uptake. N-alkane uptake by Y. lipolytica is shown to be inducible and to be inhibited by the metabolic poisons 2–4 dinitrophenol and KCN. The latter observation indicates that n-alkane uptake of Y. lipolytica is due to active transport.     

Partial cytochrome oxidase deficiency without subsarcolemmal accumulation of mitochondria in chronic progressive external ophthalmoplegia

Chronic progressive external ophthalmoplegia (CPEO) associated with proximal myopathy and/or craniosomatic abnormalities is a rare syndrome in which morphological mitochondrial changes have been found in some fibres (subsarcolemmal accumulation of mitochondria or "ragged red" fibres). We report a 14-year-old boy with CPEO and a mild proximal myopathy without these characteristic "ragged red" fibres. Histochemistry of skeletal muscle showed a mosaic of fibres without detectable cytochrome oxidase activity, while other mitochondrial enzymes were normal. The total cytochrome oxidase activity and cytochrome aa3 concentration in muscle mitochondrial fractions were only 40% of normal. This case is unique in that a biochemical defect was not accompanied by morphological abnormalities and may represent an early stage of CPEO before the development of morphological changes, or alternatively, a new variant of the disease.     

Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxygenin-labeled, polymerase chain reaction (PCR)-amplifed DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.This work was supported by the Associazione Amici del Centro Dino Ferrari     

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL~(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.     

线粒体肌病的病理分析

目的:探讨线粒体肌病患者的临床特点及其组织化学病理、电镜超微结构病理特征,提高对本病的确诊率。方法:对4例患者肌活检组织进行光镜和电镜超微病理观察。结果:3例患者肌活检组织MGT染色光镜检查发现破碎红纤维,4例患者肌活检组织电镜下发现线粒体堆积于肌原纤维间及肌膜下,形态变异,均可见晶状包涵体。结论:MGT染色发现破碎红纤维,电镜下线粒体堆积、形态异常,特别是线粒体内晶状包涵体,对本病的诊断有重要价值。     

Isolation and characterization of mutants as an approach to a transformation system in Kluyveromyces marxianus

 A method to obtain K. marxianus mutants has been developed. Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively. All his mutants analyzed by complementation tests turned out to belong to the same complementation group. Some of them were transformed and complemented by the S. cerevisiae HIS3 gene. These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry. Received: 15 November/21 December 1995     

Binding and uptake of putative neurotransmitters in mutant mouse cerebellum and cerebellar reaggregat

γ-Amino butyric acid (GABA) appears to be the inhibitory transmitter of various cerebellar interneurons, while glutamate has been suggested as the excitatory transmitter of granule cells. In this study we have determined the developmental profile from day 2 to day 60 for the Na^a-independent (presumably post-synaptic receptor) binding of [³H]GABA (K_d = 34 nM) and [³H]kainic acid, a glutamate analog (K_d = 53 nM. Both binding activities increase steadily to adult levels with kainic acid achieving this more rapidly. Cerebellar cells (7 day) cultured as reaggregates for 14 days showed an increase in GABA binding and glutamate uptake compared to the starting tissue but did not achieve the levels seen in vivo, while kainic acid binding and GABA uptake remained low. Binding and uptake measurements were performed in neurological mutants. Agranular mutants, weaver, reeler and staggerer demonstrated as expected, marked decreases in total GABA binding and glutamate uptake into synaptosomes, and to a lesser extent GABA uptake. In addition total kainate binding was depressed. In comparison, Purkinje cell degeneration mutants showed only a small decrease in kainic acid binding. These results suggest that kainic acid binding does not identify the post-synaptic glutamate receptor.     

戊型肝炎病毒变异株的血清学特征

探讨戊型肝炎病毒（ＨＥＶ）患者血清学特征。方法用逆转录聚合酶链反应法（ＲＴ－ＰＣＲ）检测ＨＥＶＲＮＡ,并对阳性产物进行克隆和测序;对部分ＨＥＶＲＮＡ阳性者进行追踪并检测抗一ＨＥＶ。结果１５例（２３.４％）为ＨＥＶＲＮＡ阳性,其中９例为典型的中国ＨＥＶ株基因序列,６例变异较大,与典型的中国株基因序列的同源性仅为８０％左右。感染原型ＨＥＶ株的４例中,３例于１月后抗一ＨＥＶ阳转;而２例感染变异ＨＥＶ株的患者于１月或３月时,抗－ＨＥＶ仍为阴性。结论现行的抗一ＨＥＶＥＩＡ试剂尚不能诊断变异的ＨＥＶ株。