Cloning and expression analysis of takeout/JHBP family genes of silkworm, Bombyx mori

A Bombyx EST cDNA database was searched using the Drosophila takeout gene and nine cDNAs were obtained. The homology search suggested that these genes are widespread in insects and organize a large gene family, and that they have hydrophobic ligands. A phylogenetic tree indicated that the genes are first divided into two large groups, juvenile hormone binding protein and other protein genes, and the latter group diversified within a short time at an early stage. The expression study of five Bombyx genes indicated that they are expressed in various tissues and are regulated by development and feeding conditions. The Bombyx genes might have roles related to the regulation of metabolism, growth or development related to nutritional conditions.     

前列腺癌相关新基因PCAG1的筛选和鉴定

目的筛选和鉴定新的前列腺癌相关基因,探讨其在前列腺癌中的表达情况,为研究其仵前列腺癌发生、发展中的作用奠定基础。方法通过生物学信息筛选,RT—PCR验证,确定前列腺癌相关新基因PCAG1。然后提取14例配对前列腺癌及癌旁组织标本中总RNA,经逆转录获得cDNA,应用RTPCR检测标本中PCAG1mRNA表达水平。免疫组织化学染色检测PCAG1在38例前列腺癌组织及配对邻近正常组织中蛋白表达水平,免疫荧光法确定PCAG1蛋白亚细胞定位,并通过统计学方法分析PCAG1表达水平与前列腺癌的关系。结果RT—PCR及免疫组化显示PCAG1在前列腺癌组织中的表达明显高于癌旁正常组织,且只在少数几种正常组织中低表达,多数正常组织中无表达,呈现明显的前列腺癌特异高表达的特性;免疫荧光确定PCAG1蛋自主要定位于线粒体中。结论PCAG1在前列腺癌组织中高表达,提示可能与前列腺癌的发生、发展相关。PCAG1独特的转录调控模式预示其具有潜在的临床应用价值。     

The tetraspanin superfamily in insects

We describe four members of the tetraspanin/TM4SF superfamily of proteins that were identified in expressed sequence tag projects on the antennae of Manduca sexta moths and Apis mellifera honey bees. The three moth genes are expressed in the sensillar epithelium of male antennae, and some are expressed in female antennae, haemocytes, wing scale cell primordia and/or embryonic tissues. These proteins are probably involved in diverse cellular processes, much like their vertebrate homologues. A phylogenetic analysis of all known tetraspanins, including thirty-seven members of the superfamily revealed by the Drosophila melanogaster genome and twenty in the nematode Caenorhabditis elegans genome, reveals some phylum-specific gene amplification, in particular a contiguous array of eighteen genes in the D. melanogaster genome.     

Cancer-testis antigens: promising targets for antigen directed antineoplastic immunotherapy

During the last decade, the aberrant expression of normal testicular proteins in neoplastically transformed cells became common knowledge. Cancer-testis antigens (CTAs) represent a novel family of immunogenic proteins. The genes MAGE, BAGE, GAGE, LAGE and NY-ESO-1 code for antigens that are recognised on various neoplastically transformed cells by autologous, cytolytic CD8 ( + ) T lymphocytes. The MAGE genes were initially analysed from melanomas and turned out to have an almost exclusively neoplasm specific expression pattern. In normal adult tissues, most 23 human MAGE genes are expressed only in the testis, with expression patterns suggesting that this gene family is involved in germ cell development. The SSX (synovial sarcoma on X chromosome) gene family, located on the X chromosome, encode a family of highly homologous nuclear proteins. A number of observations confirmed that all five SSX genes were expressed in normal testis. The newly detected CTA, NY-ESO-1, is regarded as one of the most immunogenic antigens ever isolated, inducing spontaneous host immune responses in 50% of patients with NY-ESO-1-expressing neoplasms. The identification of neoplasm-associated markers recognised by cellular or humoral effectors of the immune system has opened new perspectives for antigen directed, individualised antineoplastic immunotherapy. In preparation for this new era of targeted immunotherapy, a number of neoplasm-associated antigen families have been identified as targets for CD8+, cytolytic T lymphocytes in vitro and in vivo : (1) CTAs expressed in various neoplasms and in normal testis, restricted to male germ cells; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens overexpressed in neoplastic tissues; and (5) viral antigens. Immunotherapeutic protocols directed against the CTAs have already been initiated to analyse the induction of antigen-specific cellular and humoral immune responses in vivo.     

Drosophila yolk protein produced in E. coli is accumulated by mosquito ovaries

Despite similar functions, the yolk proteins of the higher dipteran flies and the vitellogenins found in other insects are unrelated at the sequence level and have evolved from different genes. Both are selectively endocytosed into the ovary via receptors belonging to the LDLR receptor subfamily. We cloned the Drosophila yp1 gene into an E. coli expression vector and showed that the yolk protein produced by E. coli is taken up into ovaries of both Drosophila melanogaster and the malaria mosquito Anopheles gambiae, which normally uses vitellogenin.     

Cancer-testis antigens: promising targets for antigen directed antineoplastic immunotherapy

During the last decade, the aberrant expression of normal testicular proteins in neoplastically transformed cells became common knowledge. Cancer-testis antigens (CTAs) represent a novel family of immunogenic proteins. The genes MAGE, BAGE, GAGE, LAGE and NY-ESO-1 code for antigens that are recognised on various neoplastically transformed cells by autologous, cytolytic CD8⁺ T lymphocytes. The MAGE genes were initially analysed from melanomas and turned out to have an almost exclusively neoplasm specific expression pattern. In normal adult tissues, most 23 human MAGE genes are expressed only in the testis, with expression patterns suggesting that this gene family is involved in germ cell development. The SSX (synovial sarcoma on X chromosome) gene family, located on the X chromosome, encode a family of highly homologous nuclear proteins. A number of observations confirmed that all five SSX genes were expressed in normal testis. The newly detected CTA, NY-ESO-1, is regarded as one of the most immunogenic antigens ever isolated, inducing spontaneous host immune responses in 50% of patients with NY-ESO-1-expressing neoplasms. The identification of neoplasm-associated markers recognised by cellular or humoral effectors of the immune system has opened new perspectives for antigen directed, individualised antineoplastic immunotherapy. In preparation for this new era of targeted immunotherapy, a number of neoplasm-associated antigen families have been identified as targets for CD8⁺, cytolytic T lymphocytes in vitro and in vivo: (1) CTAs expressed in various neoplasms and in normal testis, restricted to male germ cells; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens overexpressed in neoplastic tissues; and (5) viral antigens. Immunotherapeutic protocols directed against the CTAs have already been initiated to analyse the induction of antigen-specific cellular and humoral immune responses in vivo.     

Gene annotation of nuclear receptor superfamily genes in the kissing bug Rhodnius prolixus and the effects of 20-hydroxyecdysone on lipid metabolism

The hormone 20-hydroxyecdysone is fundamental for regulating moulting and metamorphosis in immature insects, and it plays a role in physiological regulation in adult insects. This hormone acts by binding and activating a receptor, the ecdysone receptor, which is part of the nuclear receptor gene superfamily. Here, we analyse the genome of the kissing bug Rhodnius prolixus to annotate the nuclear receptor superfamily genes. The R. prolixus genome displays a possible duplication of the HNF4 gene. All the analysed insect organs express most nuclear receptor genes as shown by RT-PCR. The quantitative PCR analysis showed that the RpEcR and RpUSP genes are highly expressed in the testis, while the RpHNF4-1 and RpHNF4-2 genes are more active in the fat body and ovaries and in the anterior midgut, respectively. Feeding does not induce detectable changes in the expression of these genes in the fat body. However, the expression of the RpHNF4-2 gene is always higher than that of RpHNF4-1. Treating adult females with 20-hydroxyecdysone increased the amount of triacylglycerol stored in the fat bodies by increasing their lipogenic capacity. These results indicate that 20-hydroxyecdysone acts on the lipid metabolism of adult insects, although the underlying mechanism is not clear.     

Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus

The products of the maternal-effect genes, nanos (nos) and oskar (osk), are important for the development of germ cells in insects. Furthermore, these genes have been proposed as candidates for donating functional DNA regulatory sequences for use in gene drive systems to control transmission of mosquito-borne pathogens. The nos and osk genes of the cosmopolitan vector mosquito, Culex quinquefasciatus, encode proteins with domains common to orthologues found in other mosquitoes. Expression analyses support the conclusion that the role of these genes is conserved generally among members of the nematocera. Hybridization in situ analyses reveal differences in mRNA distribution in early embryos in comparison with the cyclorraphan, Drosophila melanogaster, highlighting a possible feature in the divergence of the clades each insect represents.     

脐血干细胞向巨核细胞分化后信号转导基因的表达

背景:造血干细胞具有多向分化的潜能,在合适的造血生长因子和系特异性生长因子的诱导下,可分化成巨核细胞。在体外诱导CD34~ 造血干细胞向巨核细胞分化可导致许多基因尤其是信号转导基因表达的变化。目的:观察脐血干细胞向巨核细胞分化后信号转导基因的表达,拟从基因水平验证其对巨核细胞分化发育的调控。设计:开放性实验。单位:福建省肿瘤医院肿瘤免疫学研究室。材料:无菌条件下应用血袋采集法收集足月正常分娩的胎儿脐带血60～145 mL,产妇及家属自愿捐献。VarioMACS免疫磁性吸附柱分离装置,CD34 Isolation Kit;SCGM无血清培养基;CD单抗;细胞因子。人类全基因组寡核苷酸微阵列芯片V2.0;LuxScan 10K/A双通道激光扫描仪。实验经医院伦理委员会批准。方法:实验于2005-07/2006-06在福建省肿瘤医院肿瘤免疫研究室及北京博奥生物芯片有限公司完成。分别收集同一份脐血分化培养前CD34~ 细胞和培养后CD41~ 细胞,提取总RNA。逆转录合成和纯化cDNA探针,以Cy3-dCTP标记培养前CD34~ 细胞,以Cy5-dCTP标记培养后CD41~ 细胞标记的DNA溶于30μL杂交液中,42℃过夜。主要观察指标:应用基因芯片技术比较造血干细胞与分化的巨核细胞信号转导相关的基因表达差异。根据基因芯片结果,选定17个表达差异基因用RT-PCR作进一步验证。结果:芯片分析结果显示,共筛选出3522个差异基因,其中上调基因1705个,下调基因1817个。3522个差异基因中,与细胞信号相关的基因有343个,与转录调节相关的有150个,与分化相关的有21个。其中,CD61基因的表达增加了369.83倍,CD41基因的表达增加27.38倍,PF4基因的表达增加24.06倍;促分裂原活化蛋白澈酶s、G蛋白偶联受体、RAS家族相关的基因多数表达上调;与STAT通路相关的基因中,SOCS1、JANUS激酶表达上调,STAT5A表达下调。对选定的17个表达差异基因用RT-PCR作进一步验证,以GAPDH为内对照,结果与芯片检测完全一致。结论:血小板生成素等造血生长因子可能主要通过G蛋白偶联受体-Ras-促分裂原活化蛋白激酶途径,促进脐血干细胞向巨核细胞系增殖、分化。