Mapping of sporulation-specific functions in the yeast syntaxin gene<Emphasis Type="Italic"> SSO1</Emphasis>

The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells. However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p. We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1. We found that the two N-terminal -helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p. In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation. Surprisingly, we further found that the 3 untranslated region (3UTR) of SSO1 is essential for sporulation. Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3UTR is needed for sporulation.Communicated by S. Hohmann     

Day-night differences in estimated rates of 5-hydroxytryptamine turnover in the rat pineal gland

Summary Rates of 5-hydroxytryptamine synthesis in various brain tissues can be estimated from the linear increase in 5-hydroxytryptophan levels following inhibition of 5-hydroxytryptophan decarboxylation with RO4-4602 or NSD-1015. In addition, NSD-1015 can prevent 5-hydroxytryptamine oxidative-deamination via monoamine oxidase inhibition, leading to linear decreases in a major metabolite of this amine, 5-hydroxyindole acetic acid. In the rat pineal gland we demonstrated similar increases in 5-hydroxytryptophan levels after nocturnal or diurnal injection of RO4-4602 (100 mg · kg^–1) or NSD-1015 (200 mg · kg^–1). Similar decreases in 5-hydroxyindole acetic acid were also observed after nocturnal or diurnal injection of NSD-1015 or pargyline (an inhibitor of monoamine oxidase) (75 mg · kg^–1). 5-Hydroxytryptamine levels increased after nocturnal pargyline injection but remained constant after diurnal pargyline administration. 5-Hydroxytryptamine levels exhibited little change following nocturnal injection of NSD-1015 but decreased linearly after diurnal injection of NSD-1015. We suggest that (1) rat pineal 5-hydroxytryptamine synthesis is increased nocturnally, (2) metabolic utilization, primarily by oxidative-deamination, of 5-hydroxytryptamine is increased diurnally and (3) basal levels of pineal 5-hydroxytryptamine may be stored within adrenergic nerve endings which innervate the pinealocytes responsible for synthesizing this amine, thus protecting or otherwise making unavailable this pool of 5-hydroxytryptamine for metabolic utilization.     

Analysis of T cell repertoire in the liver of patients with chronic hepatitis C

Many T cells infiltrate into the liver of patients with chronic hepatitis C (CH-C). They are believed to play a crucial role in the immunopathogenesis of hepatic inflammation, but their clonality and specificity are unknown. The aim of this study was to clarify the characteristics of these T cells. We analysed the complementarity-determining region (CDR)3 size lengths of T cell receptor (TCR) beta-chains by size spectratyping, and determined the sequences of Vbeta CDR3 after subcloning Vbeta-specific polymerase chain reaction products. Spectratyping showed clonal expansions in all liver specimens, most of which showed more than two T cell clones. Moreover, many non-clonal T cells also accumulated in the liver. Clonality of the T cells suspected by spectratyping was confirmed by CDR3 sequencing. Although the sequences revealed no whole CDR3-shared clones among different patients, some common motif sequences were observed. Our data suggest that T cells are stimulated by several hepatitis C virus (HCV) epitopes, then accumulate in the liver of CH-C patients. Shared motifs of expanded T cell clones suggest that they might recognize the same regions of HCV peptides, but have differences due to HCV peptide mutational changes. These clones might also interact with non-clonal T cells and play a crucial role in the immunopathogenesis of CH-C.     

塞姆利基森林病毒翻译增强序列对HIV Gag DNA疫苗抗原表达和免疫原性的调节

目的 研究塞姆利基森林病毒（Semliki forest virus，SFV）衣壳蛋白5’翻译增强区对基于该病毒复制子的HIV Gag DNA疫苗抗原表达水平和免疫原性的影响：方法将SFV衣壳蛋白（capsid，C蛋白）基因的5’端102bp翻译增强区插入到SFT复制子DNA疫苗载体pCMV-Rep的SFV亚基因启动子下游，得到DNA疫苗载体pCMV-RepC。将删除ATG的HIV-1 gag基因插入pCMV-RepC，使gag编码区与翻详增强区融合，得到DNA疫苗质粒pCMV-RepC-gag。同时，构建携带未融合翻译增强序列的DNA疫苗质粒pCMV-Rep-gag。Western blot检测翻译增强序列对Gag表达水平的影响。用上述两种DNA疫苗分别免疫BALB／c雌性小鼠，ELISA检测Gag特片的抗体反应，ELISPOT和细胞内因子染色技术检测细胞免疫应答。结果衣壳蛋白5’翻译增强区增强了Gag表达水平，对体液免疫应答没有显著影响，但显著增强了特异性细胞免疫应答水平。结论SFVC蛋白翻译增强区能显著提高SFV复制子DNA疫苗的抗原表达和抗原特异性细胞免疫反心。     

Comparison of anatomical location of squamous cell carcinoma within the oral cavity and oropharynx with the incidence of in vitro hyperdiploidy

The anatomical location of the squamous cell carcinoma (SCCA) within the oral cavity and oropharynx influenced the association of SCCA with the biomarker in vitro hyperdiploidy in human dermal fibroblast cultures (IVH). There was a strong association of IVH with the occurrence of SCCA in the anterior 2/3 of the tongue, floor of the mouth and lower alveolar ridge of the oral cavity and in the base of the tongue and pharyngeal wall of the oropharynx. There was a lower association of SCCA with IVH in the tonsillar region of the oropharynx. IVH showed no association with SCCA located in other anatomical parts of the oral region. The patient group whose diagnosis of SCCA in the anterior 2/3 of the tongue occurred prior to the age of 50 years were invariably IVH-, whereas those diagnosed after the age of 50 years were IVH+, providing evidence for heterogeneity. There was no such correlation of biomarker subgrouping with age of diagnosis demonstrated for SCCA at any other anatomical location within the oral cavity or oropharynx.     

Selective cytotoxic lesions of the retrohippocampal region produce a mild deficit in social recognition memory

Although a number of studies have implicated the hippocampal formation in social recognition memory in the rat, a recent study in this laboratory has demonstrated that selective cytotoxic lesions, confined to the hippocampus proper (encompassing the four CA subfields and the dentate gyrus), are without effect on this behaviour. This finding suggests that the hippocampus proper does not subserve social recognition memory in the rat, but does not preclude the possibility that other areas of the hippocampal formation, such as the entorhinal cortex or subiculum, could support this form of learning. The present study addressed this issue by examining the effects of selective cytotoxic retrohippocampal (RHR) lesions (including both the entorhinal cortex and subiculum) on social recognition memory in the rat. RHR lesions produced a mild social recognition memory impairment, although lesioned animals still displayed a reduction in investigation time between the first and second exposure to the juvenile. This result is consistent with other studies which have implicated the retrohippocampal or parahippocampal area in olfactory recognition memory processes. It also suggests, however, that other areas, out with the retrohippocampal region, are also likely to play an important role in social recognition memory.     

The haplotype block, NFKBIL1-ATP6V1G2-BAT1-MICB-MICA, within the class III-class I boundary region of the human major histocompatibility complex may control susceptibility to hepatitis C virus-associated dilated cardiomyopathy

Cardiomyopathy is a heart muscle disease with impaired stretch response that can result in severe heart failure and sudden death. A small proportion of hepatitis C virus (HCV)-infected patients may be predisposed to develop dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). The molecular mechanisms involved in the predisposition remain unknown due in part to the lack of information on their genetic background. Because the human leukocyte antigen (HLA) region has a pivotal role in controlling the susceptibility to HCV-induced liver disease, we hypothesized that particular HLA alleles and/or non-HLA gene alleles within the human major histocompatibility complex (MHC) genomic region might control the predisposition to HCV-associated DCM (HCV-DCM) and/or HCV-associated HCM (HCV-HCM). Here, we present mapping results of the MHC-related susceptibility gene locus for HCV-associated cardiomyopathy by analyzing microsatellite and single nucleotide polymorphism markers. To delineate the susceptibility locus, we genotyped 44 polymorphic markers scattered across the entire MHC region in a total of 59 patients (21 HCV-DCM and 38 HCV-HCM) and 120 controls. We mapped HCV-DCM susceptibility to a non-HLA gene locus spanning from NFKBIL1 to MICA gene loci within the MHC class III-class I boundary region. Our results showed that HCV-DCM was more strongly associated with alleles of the non-HLA genes rather than the HLA genes themselves. In addition, no significant association was found between the MHC markers and HCV-HCM. This marked difference in the MHC-related disease susceptibility for HCV- associated cardiomyopathy strongly suggests that the development of HCV- DCM and HCV-HCM is under the control of different pathogenic mechanisms.     

9.1C3单克隆抗体轻,重链可变区基因克隆及序列分析

９．１Ｃ３分子是一种参与ＮＫ、巨噬细胞及ＬＡＫ细胞杀伤过程的重要细胞表面分子，主要表达于外周血单个核细胞表面。本文运用分子生物学技术，从分泌９．１Ｃ３ＭＣＡｂ的杂交瘤细胞中提取总ＲＮＡ，经反转录、ＰＣＲ分另１１分离了９．１Ｃ３ＭｃＡｂ轻链可变区（ＶＬ）基因及重链可变区（ＶＨ）基因，并用全自动荧光ＤＮＡ序列分析仪对９．１Ｃ３ＶＨ、ＶＬ基因序列进行了分析。结果表明：９．１Ｃ３ＶＨ基因为３５１ｂＰ，编码１１７ａａ残基，９．１３ＶＬ为３２４ｂＰ，编码１０８ａａ残基；从推导出的氨基酸序列分析证实９．１Ｃ３ＶＨ、ＶＬ序列均符合小鼠ｌｇ可变区的氨基酸序列特征；根据Ｋａｂｂａｔ分类体系，９．１Ｃ３ＶＨ隶属Ｉｇ重链第Ⅱ（Ｃ）亚组，９．１Ｃ３ＶＬ隶属小鼠ＩｇＫ链第Ⅴ亚组。９．１Ｃ３ＶＨ、ＶＬ基因的克隆成功为其单链抗体的构建、表达奠定了良好的物质基础。