Expression and immunolocalization of a Boophilus microplus cathepsin L-like enzyme

Efforts are being undertaken to control tick infestations that cause important economic losses. A cathepsin L-like endopeptidase of Boophilus microplus was expressed in Escherichia coli; the recombinant enzyme was capable of hydrolysing gelatin, tick vitellin and bovine haemoglobin. In this paper we focus on the expression and local of synthesis of this enzyme in the tick. RT-PCR experiments showed that this endopeptidase is transcribed in the gut of partially engorged tick females. In immunoblotting, polyclonal antibodies against the recombinant enzyme reacted with proteins of larvae older than 5 days, of fully and partially engorged female gut. In immunolocalization experiments the enzyme was localized in probable secretory cells of the gut. Based on our findings we postulate that BmCL1 may be involved in haemoglobin degradation in the B. microplus gut. This enzyme may be used as target for the control of this parasite.     

Genome analysis with gene-indexing databases.

The recent release of the draft sequence and the eventual completion of the human genome present the scientific community with a rich source of data to mine. Yet, these data are content poor in the absence of additional correlative information. Expressed sequence tag (EST) datasets and their associated gene indices have existed for many years, and represent the first attempt at understanding the complexity of the genome. These datasets remain extremely important as information sources and, in particular, as tools for analyzing the completed genomes. Here, we discuss the nature of ESTs and their associated tools and gene-indexing databases. In particular, we will compare three EST gene indices (UNIGENE, Merck Gene Index Version 2.0 and Doubletwist CAT), discuss how these gene indices are applied for both genome analysis and drug discovery, and demonstrate their importance as a complementary dataset to the annotated human genome.     

Genes related to immunity, as expressed in the alfalfa leafcutting bee, Megachile rotundata, during pathogen challenge

Virtually nothing is known about disease resistance in solitary bees, so expressed sequence tag (EST) databases were developed to search for immune response genes in the alfalfa leafcutting bee. We identified 104 putative immunity-related genes from both healthy and pathogen-challenged bee larvae, and 12 more genes using PCR amplification. The genes identified coded for proteins with a wide variety of innate immune response functions, including pathogen recognition, phagocytosis, the prophenoloxidase cascade, melanization, coagulation and several signalling pathways. Some immune response genes were highly conserved with honey bee genes, and more distantly related to other insects. The data presented provides the first analysis of immune function in a solitary bee and provides a foundation for the further analysis of gene expression patterns in bees.     

Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae)

The midgut is a key tissue in insect science. Physiological roles include digestion and peritrophic membrane function, as well as being an important target for insecticides. We used an expressed sequence tag (EST) approach to identify candidate genes and gene families involved in these processes in the light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae). Two cDNA libraries were constructed from dissected midgut of third to fifth instar larvae. Clustering analysis of 6416 expressed sequence tags produced 1178 tentative unique genes comprising 725 tentative contigs and 453 singletons. The sequences show similar codon usage to sequences from other lepidopterans, a Kozak consensus sequence similar to Drosophila and single nucleotide polymorphisms (SNPs) were detected at a frequency of 1.35/kb. The identity of the most common Interpro families correlates well with major known functions of the midgut. Phylogenetic analysis was conducted on representative sequences from selected multigene families. Gene families include a broad range of digestive proteases, lipases and carbohydrases that appear to have degradative capacity against the major food components found in leaves, the diet of these larvae; and carboxylesterases, glutathione-S-transferases and cytochrome P450 monooxygenases, potentially involved in xenobiotic degradation. Two of the larger multigene families, serine proteases and lipases, expressed a high proportion of genes that are likely to be catalytically inactive.     

Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure

A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families.     

Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over‐expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory‐selected OP‐resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory‐selected cotton aphid strain.     

人直肠腺癌消减杂交差异片段的生物信息学分析

目的探测人直肠腺癌发生和发展过程中差异表达的基因片段,了解人直肠腺癌的分子生物学机制。方法应用抑制性消减杂交技术,获得人直肠腺癌差异表达的基因,用生物信息学cDNA文库筛选的方法,对人直肠腺癌cDNA消减文库中的差异基因片段的序列作预测和分析。结果用抑制性消减杂交技术随机挑取获得的阳性克隆,提取质粒,双酶切分析,进行序列测定,得到与直肠腺癌发病相关的差异基因片段-9cDNA(Genbank登录号为BM360862),用cDNA文库筛选得到一个全长cDNA序列。基因表达系列分析(SAGE)显示除了直肠腺癌外、9cDNA还分布于前列腺腺癌、脑干成神经管细胞瘤、胰腺腺癌、胚胎正常血管内皮细胞、脑室管细胞瘤。结论直肠腺癌的发病是多环节和多步骤的过程,9cDNA与直肠腺癌发病相关,对9cDNA基因差异片段进一步研究,将为了解直肠腺癌发病的特异相关基因奠定基础。     

抑制性差减杂交技术分离白血病多药耐药基因

目的分离及鉴定与白血病多药耐药性相关的差异表达基因。方法采用抑制性差减杂交(SSH)技术分离非耐药细胞株K562与耐药细胞株K562/DOX差异表达基因。提取总RNA,逆转录合成cDNA,经限制性内切酶RsaⅠ酶切后,分别与不同的接头(adopter1和adopter2R)连接;连接产物插入pMD19-T载体后转入大肠埃希菌中,构建cDNA差减文库;挑取阳性克隆提取质粒进行测序及同源序列分析,确定差异表达基因。结果筛选获得220个差异表达基因,包括血红蛋白、核糖体和线粒体等相关基因,以及热休克因子结合蛋白(HSPB1)基因等其他基因。结论采用SSH技术及分子克隆技术可构建耐药及非耐药肿瘤细胞株差异表达基因的差减cDNA文库,能够为进一步筛选、克隆肿瘤细胞多药耐药性相关差异表达基因奠定基础。     

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.     

A viral histone H4 suppresses expression of a transferrin that plays a role in the immune response of the diamondback moth,Plutella xylostella

A transferrin (Tf) gene has been predicted from an expressed sequence tag of the diamondback moth, Plutella xylostella. It encodes 681 amino acid residues that share 80–90% sequence homologies with other lepidopteran Tfs. The gene was constitutively expressed in all developmental stages of P. xylostella. Double‐stranded RNA (dsRNA) specific to the Tf gene was prepared and microinjected into the larvae. We hypothesize that the dsRNA treatment suppressed the Tf gene expression level and it significantly inhibited haemocyte nodule formation in response to bacterial challenge. The larvae treated with dsRNA also showed a significantly enhanced susceptibility to an entomopathogenic bacterium, Bacillus thuringiensis. An endoparasitoid wasp, Cotesia plutellae, parasitized the larvae of P. xylostella, which showed significant reduction of Tf expression. The suppression of Tf expression was mimicked by transient expression of a viral gene CpBV‐H4, encoded in the symbiotic virus of C. plutellae. A truncated form of CpBV‐H4 prepared by deleting an extended N‐terminal 38 amino acid residue lost its inhibitory activity against the Tf gene expression. These results suggest that Tf of P. xylostella plays an immunological role in P. xylostella and that the suppression of its expression in the parasitized larvae is caused by a viral histone H4 in an epigenetic mode.     

The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae)

We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.     

Comparative proteome analysis of silkworm in its susceptibility and resistance responses to Bombyx mori densonucleosis virus

Bombyx mori densonucleosis virus (BmDNV) is one of the most disastrous viruses in cocoon production. Silkworm resistance to BmDNV has been examined previously using a number of traditional biochemical and molecular techniques. In this study, a near isogenic line, BC(6), was constructed to eliminate the difference in inherited background, which has 99.9% identity with the susceptible strain but carries a resistant gene. We utilized a proteomic approach involving two-dimensional differential gel electrophoresis and mass spectrometry to examine changes in the midgut proteins from the susceptible and resistant silkworm larvae infected with BmDNV. The protein profiles were compared and 9 differentially expressed proteins were identified by mass spectrometry. In the resistant strains, the heat-shock 70-kDa protein cognate, cytochrome P450, vacuolar ATP synthase subunit B, arginine kinase, vacuolar ATP synthase subunit D and glutathione S-transferase sigma were strongly upregulated and α-tubulin was downregulated. Our results imply that these upregulated genes and the downregulated genes might be involved in B. mori immune responses against BmDNV-Z infection.     

2型糖尿病家族史风险通路识别及功能分析

目的: 探讨2型糖尿病家族史与胰岛素抵抗间的关系,采用信息学方法对从基因表达库（Gene Expression Omnibus, GEO）获取的具有2型糖尿病家族史但自身血糖正常的研究对象进行相关基因表达谱分析,识别2型糖尿病疾病风险通路,并对关键基因产物进行功能分析。方法: 对美国国立生物技术信息中心（National Center for Biotechnology Information,NCBI）的综合性基因表达与杂交阵列数据库中26名有2型糖尿病家族史但自身血糖正常的研究对象和15名无糖尿病家族史的血糖正常的人群进行数据分析（GSE25462）,运用Bergman微小模型技术结合静脉糖耐量试验评估胰岛素敏感指数,并对骨骼肌细胞基因表达谱芯片采用生物信息学方法（基因功能富集分析方法、风险通路识别方法等）进行数据分析,进而在分子层面进行2型糖尿病致病机制研究。结果: 有2型糖尿病家族史但自身血糖正常的研究对象其胰岛素敏感性下降41%（P<0.05）。通过差异表达分析,共发现202个基因表达在家族史阳性组与家族史阴性组间的差异有统计学意义,同时鉴定了富集差异显著的5条通路,分别是多能干细胞的造血功能、万能干细胞的造血功能、JAK1/3在c-细胞活素信号通路的作用、TOB在T细胞信号通路中的抗增殖作用、B细胞的发育。结论: 有2型糖尿病家族史但自身血糖正常的研究对象存在胰岛素抵抗,2型糖尿病的致病机制与造血干细胞功能及免疫状态有重要联系。     

Genetically determined, interferon-dependent resistance to influenza virus in mice

The genetically determined resistance towards orthomyxoviruses exhibited by mice homozygous (A2G) or heterozygous (A2G X A/J) for the gene Mx was abolished or greatly diminished by treatment with anti-interferon globulin (AIF). AIF induced increased susceptibility to challenge with hepatotropic, neurotropic, and pneumotropic strains of influenza A virus. Hepatotropic virus titers in blood and livers of AIF-treated, Mx-bearing mice were higher by a factor of 10(3)--10(6) than those in untreated mice of the same genotype, and were comparable to those in genetically susceptible (untreated or AIF-treated) mice. Peritoneal macrophages from Mx-bearing untreated mice were resistant to challenge with a macrophage-adapted strain of influenza A virus even in the presence of AIF. However, when macrophages were taken from resistant mice injected with AIF and also cultivated in the presence of AIF, they were as susceptible to the virus as macrophages taken from susceptible mice. We conclude that interferons is an important factor in resistance to orthomyxoviruses governed by the gene Mx.