Identification of a novel S-superfamily conotoxin from vermivorous Conus caracteristicus

Conotoxins have been classified into several different superfamilies based on the highly conserved signal peptide sequences of their precursors. However, little is known about the five disulfide bonds containing S-superfamily conotoxins. Only two S-superfamily conotoxins have been identified but their cDNAs are not reported. In this work, we identified a novel S-superfamily conotoxin ca8a from vermivorous Conus caracteristicus. Its sequence shares no homology with those of two other previously reported toxins of the same superfamily, but they have the same cysteine framework, in particular the CX(3)CXC-CXC-CXCXC pattern at the C-terminal part. This implies that these toxins might have the same spatial scaffold, but different local conformation or residue side chains may be the cause of their different biological functions. Furthermore, the cDNA of ca8a was cloned with the RACE method. ca8a has a signal peptide sequence different from those of other conotoxins. This gives a defining feature of S-superfamily conotoxins and led to the cloning of more S-superfamily conotoxins from cone snails of different prey types, which indicates that S-superfamily conotoxins widely exist. These results will certainly enrich our understanding of the highly diversified S-superfamily conotoxins.     

Nucleotide sequences of putative cDNAs for guinea-pig monoamine oxidase

To study the molecular structure of guinea pig monoamine oxidase (MAO) and its phylogenetic relationship with other mammalian MAOs, we determined nucleotide sequences of putative MAO cDNAs isolated from guinea pig tissues. Both the 5- and 3-ends of the cDNAs were amplified using the RACE (rapid amplification of cDNA ends) method. The sequence (1924 bp) of a putative guinea-pig MAO-B cDNA covers a complete coding region that corresponds to 521 amino acids. We also analyzed a partial sequence of a putative guinea-pig MAO-A cDNA, which corresponds to 506 amino acids, but have left the region of 66 bp at the 3-end undetermined. The nucleotide and deduced amino-acid sequences of the putative guinea-pig MAO cDNAs showed the highest homology with that of human MAO cDNAs among the known mammalian MAO sequences. These results suggest that guinea-pig MAOs are structurally similar to human MAOs. Our molecular phylogenetic data support the idea that guinea pigs and rodents diverged before the separation between rodents and other lineage leading to Primates and Artiodactyla.     

Drug transporter expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line

Ovarian cancer is characterized by the higher mortality among gynecological cancers. In results of MDR development during chemotherapy cancer cells become resistant to further treatment. Microarray techniques can provide information about MDR development at gene expression level. ABC and SLC transporters are most important proteins responsible for this phenomenon. In this study changes of ABC and SLC genes expression pattern in drugs resistant sublines of the A2780 ovarian cancer cell line were demonstrated. The cytostatic resistant sublines were generated by culture of A2780 cell line with an increasing concentration of the indicated drugs. As screening methods, we used Affymetrix U219 Human Genome microarrays. Independent t-tests were used to determinate statistical significances of results. Genes that expression levels were higher than assumed threshold (upregulated above threefold and downregulated under −3 fold) were visualized using scatter plot method, selected and listed in table. Between the ABC genes increased expression of seven genes and decreased expression of three genes were observed. Expression of two genes was increased or decreased depending on the cell line. We observed significant (more than tenfold) increase in expression of four ABC genes: ABCA8, ABCB1, ABCB4 and ABCG2 and decreased expression of ABCA3 gene. We also observed changes in expression of 32 SLC genes. Between them we observe increased expression of 17 genes and decreased expression of 15 genes. Expression of four genes was increased or decreased dependent on cell line. The expression of nine SLC genes increased or decreased very significantly (more than tenfold). Five genes were significantly upregulated: SLC2A9, SLC16A3, SLC16A14, SLC38A4 and SLC39A8. Four additional genes were significantly downregulated: SLC2A14, SLC6A15, SLC8A1 and SLC27A2. Expression profiles of these genes give strong arguments for assumption of correlation between expression of ABC and SLC genes and drug resistance phenomenon. Identifying correlations between specific drug transporters and cytostatic drug resistance will require further investigation.     

虚寒证-基因组预选的代谢类基因功能模块

目的 对课题组近年探索虚寒证-基因组的研究进行概述,展示预选的代谢类基因功能模块.方法 在万人流行病学调查与典型虚寒证患者筛选的背景中,通过病证结合、普查辨证、家系分析、温针反证、温肾反证、动物实验等6种研究角度作基因表达谱芯片实验,并对结果进行分析.结果 虚寒证的差异表达基因较为集中地出现于代谢与免疫两类基因,对这一趋势初步总结出虚寒证的代谢类候选基因,即LPL脂蛋白脂肪酶1等35条基因.结论 虚寒证的发生涉及多种基因的异常表达,候选基因只是极其有限的第一步探索,对预选的代谢类基因功能模块的深入研究将有助于揭示寒证的本质.     

应用抑制性差减杂交技术构建铁皮石斛差减cDNA文库的探讨

【目的】探讨构建铁皮石斛抑制差减文库的方法,为克隆石斛碱生物合成相关功能基因奠定基础。【方法】采用抑制性差减杂交(SSH)技术,分别以4年生和1年生铁皮石斛叶片互补脱氧核糖核酸(cDNA)作为检测子(tester)与驱赶子(driver),将所获得差减cDNA片段克隆入质粒表达载体(Point^TMXa-1 T-Vector),转化大肠杆菌JM109,聚合酶链反应(PCR)扩增鉴定插入片段。【结果】成功地构建了与石斛碱生物合成相关的差减cDNA文库,获得的正、反向差减文库分别含560、220个重组子;插入片段的平均大小为550bp。【结论】所构建的差减cDNA文库适合进一步克隆分析石斛碱相关功能基因研究。     

MHC in a monogamous lizard – Characterization of class I MHC genes in the Australian skink Tiliqua rugosa

The major histocompatibility complex (MHC) is a highly variable region of vertebrate genomes that encodes cellular proteins involved in the immune response. In addition to the benefits of MHC research in understanding the genetic basis of host resistance to disease, the MHC is an ideal candidate for studying genetic diversity under strong natural selection. However, the MHC of many non-model vertebrate taxa are poorly characterized, hindering an understanding of disease resistance and its application to conservation genetics in these groups. Squamates (lizards and snakes) remain particularly underrepresented despite their being the most diverse order of non-avian sauropsids. We characterized MHC class I sequence diversity from an Australian skink, the sleepy lizard (Tiliqua rugosa), using both cDNA and genomic sequence data and also present genomic class I sequences from the related skinks Tiliqua adelaidensis and Egernia stokesii. Phylogenetic analysis of Tiliqua and other published sqamate MHC class I sequences suggest that MHC diverged very early in Tiliqua compared with the other studied squamates. We identified at least 4 classical MHC class I loci in T. rugosa and also shared polymorphism among T. rugosa, T. adelaidensis and E. stokesii in the sequences encoding peptide-binding α1 and α2 domains.     

The potential cost-effectiveness of infant pneumococcal vaccines in Australia

Over the last decade infant pneumococcal vaccination has been adopted as part of routine immunisation schedules in many developed countries. Although highly successful in many settings such as Australia and the United States, rapid serotype replacement has occurred in some European countries. Recently two pneumococcal conjugate vaccines (PCVs) with extended serotype coverage have been licensed for use, a 10-valent (PHiD-CV) and a 13-valent (PCV-13) vaccine, and offer potential replacements for the existing vaccine (PCV-7) in Australia. To evaluate the cost-effectiveness of PCV programs we developed a static, deterministic state-transition model. The perspective for costs included those to the government and healthcare system. When compared to current practice (PCV-7) both vaccines offered potential benefits, with those estimated for PHiD-CV due primarily to prevention of otitis media and PCV-13 due to a further reduction in invasive disease in Australia. At equivalent total cost to vaccinate an infant, compared to no PCV the base-case cost per QALY saved were estimated at A$64,900 (current practice, PCV-7; 3 + 0), A$50,200 (PHiD-CV; 3 + 1) and A$55,300 (PCV-13; 3 + 0), respectively. However, assumptions regarding herd protection, serotype protection, otitis media efficacy, and vaccination cost changed the relative cost-effectiveness of alternative PCV programs. The high proportion of current invasive disease caused by serotype 19A (as included in PCV-13) may be a decisive factor in determining vaccine policy in Australia.     

大鼠激素性股骨头坏死骨代谢差异表达基因的实验研究

目的:运用基因芯片技术,研究激素性股骨头坏死骨代谢相关基因的差异表达情况,进一步探讨其骨质疏松学说发病机理的分子机制.方法:wistar大鼠20只,随机分为对照组(n=10)和模型组(n=10),利用内毒素和甲强龙诱导激素性股骨头坏死模型,提取股骨头组织总RNA,采用基因芯片检测骨代谢关基因的表达改变.结果:基因芯片筛...     

肾上腺腺瘤相关基因的筛选及生物信息学分析

目的根据高通量基因表达谱数据,采用数据挖掘技术识别肾上腺腺瘤相关的特征基因与功能,进一步探讨G-蛋白偶联受体（GPCR）在肾上腺腺瘤中的表达情况,为临床药物治疗肾上腺腺瘤提供了依据。方法应用GeneSifter在线分析软件对5个正常肾上腺（NA）和10个肾上腺腺瘤（APA）基因芯片数据分成两组进行分析。结果筛选得到2370个差异表达基因,其中G-蛋白偶联受体41个,其中38个上调,3个下调。MC2R和HTR4已经被确认在APA病人中过表达,并在临床中作为药物作用靶点应用于治疗APA。而谷氨酸受体和GPR37还没有人研究它们在肾上腺中的作用机制。结论APA中醛固酮的生成与过表达的GPCR有潜在的关系,这些GPCR还需要被进一步研究。