山溪鲵皮肤β-microseminoprotein样蛋白的分离纯化及其cDNA克隆

从山溪鲵(Batrachuperus pachunii)皮肤匀浆液中经过Sephadex G-50凝胶过滤、AKTA(R)Resource Q阴离子柱和反向高压液相C4柱分离纯化得到相对分子质量为12 000的蛋白.利用其N端氨基酸序列设计引物,从山溪鲵皮肤的cDNA中克隆并筛选到该蛋白的cDNA序列.该cDNA序列的开放阅读框为339 bp,编码113个氨基酸残基组成的蛋白.在BLAST数据库搜寻比对分析表明,该蛋白的氨基酸序列与来自人类及其他哺乳动物β-microseminoprotein蛋白具有约40%的序列同源性.这也是首次在两栖类动物皮肤中确认β-microseminoprotein.初级结构分析表明,该蛋白属于亲水性蛋白;多重序列比较显示,其氨基酸序列中的10个半胱氨酸位点及15个其他氨基酸位点与高等脊椎动物β-microseminoprotein中同种氨基酸有相同的位点.由此推测该蛋白属于β-microseminoprotein家族.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Construction and characterization of a fosmid library for pathogenic bacterium Vibrio anguillarum

Vibrio anguillarum is a common bacterial pathogen in fish. However, little is known about its pathogenic mechanism, in part, because the entire genome has not been completely sequenced. We constructed a fosmid library for V. anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage. We characterized the library by end-sequencing 50 randomly selected clones. This generated 93 sequences with a total length of 57 485 by covering 1.4% of the whole genome. Of these sequences, 58 (62.4%) were homologous to known genes, 30 (32.3%) were genes with hypothetical functions, and the remaining 5 (5.3%) were unknown genes. We demonstrated the utility of this library by PCR screening of 10 genes. This resulted in an average of 6.2 fosmid clones per screening. This fosmid library offers a new tool for gene screening and cloning of V. anguillarum, and for comparative genomic studies among Vibrio species.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 51 untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepato-pancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge. (C) 2009 Elsevier Ltd. All rights reserved.

雨生红球藻Rubisco在虾青素积累过程中的表达分析

虾青素因其具有极强的抗氧化性及优越的着色作用，被广泛应用于营养保健和水产养殖中，备受国内外研究者的关注。红球藻是目前虾青素的生物来源中较有优势的一条途径。 我们选取雨生红球藻作为研究材料，收集其生长过程中四个不同的阶段，分别为绿色游动细胞阶段，绿色不动细胞阶段，绿褐色不动细胞阶段（开始积累虾青素），以及红色不动孢子阶段。本研究中提取了核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）的粗提液，并测定了酶活。其编码基因rbcL的mRNA表达量也被测定。另外，光合速率与呼吸速率的比值（P/R）通过测定与计算得出，各个阶段藻细胞中虾青素的含量由分析得出。本研究还应用了叶绿素荧光测定方法，确定了光系统II潜在最大活性（Fv / Fm），光系统II实际活性（ΦPSII），电子传递速率（ETR）和非光化学淬灭参数（NPQ）。 结果表明，绿色游动细胞的生长状态最佳，其P/R、Fv / Fm、ΦPSII均为最大，NPQ为最小。这说明在此状态的细胞中，光系统II的活性最强；但是其Rubisco活性与rbcL表达量均为最小。相比之下，在绿褐色不动细胞中，P/R和NPQ的值较低，Fv / Fm、ΦPSII和ETR值都最小，但Rubisco活性与rbcL表达量均为最高。 结合工业生产虾青素的方法，我们认为，Rubisco或许参与了虾青素的合成，而非Calvin循环为色素合成提供前体和能量。因此，在生产过程中适当加入碳源，比如CO2，可以有效增大虾青素的产量。

Regulacja karboksylazy/oksygenazy rybulozo-1,5-bisfosforanu (Rubisco) u Arabidopsis thaliana w odpowiedzi na zmianę natężenia światła z umiarkowanego na niskie

Wydział Biologii

The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.

Kassinakinin S: A novel histamine-releasing heptadecapeptide from frog (Kassina senegalensis) skin secretion

Amphibian defensive skin secretions remain a largely untapped resource for the peptide biochemist with an interest in the identification, structural characterization, and precursor cDNA cloning of novel bioactive peptides. Here we report the isolation, structural characterization, functional profiling, and nucleotide sequence of precursor cDNA of a novel histamine-releasing heptadecapeptide, FIPVTLLALHKIKEKLN-amide, from the defensive skin secretion of the African running frog, Kassina senegalensis. This peptide was found to be a potent histamine secretagogue (EC[5][0]=6 µM; maximal release = 25 µM) in a rat peritoneal mast cell model system and was accordingly named kassinakinin S. The open-reading frame of the cDNA encoding prepro-kassinakinin S was found to consist of 71 amino acid residues containing a single copy of kassinakinin S and its glycyl residue amide donor at the C-terminus. Kassinakinin S can thus be added to the growing list of amphibian skin bioactive peptide prototypes.

Ten years of protein kinase B signalling: a hard Akt to follow

It is ten years since the publication of three papers describing the cloning of a new proto-oncogene serine/threonine kinase termed protein kinase B (PKB)/Akt. Key roles for this protein kinase in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration are now well established. The explosion of publications involving PKB/Akt in the past three years emphasizes the high level of current interest in this signalling molecule. This review focuses on tracing the characterization of this kinase, through the elucidation of its mechanism of regulation, to its role in regulating physiological and pathophysiological processes,to our current understanding of the biology of PKB/Akt, and prospects for the future.

Ranachensinin: A Novel Aliphatic Tachykinin from the Skin Secretion of the Chinese Brown Frog, Rana chensinensis

Amphibian skin secretions contain a plethora of pharmacologically-active substances and represent established sources of bioactive peptides, including tachykinins. Tachykinins are one of the most widely-studied peptide families in animals and are found in neuroendocrine tissues from the lowest vertebrates to mammals. They are characterized by the presence of a highly-conserved C-terminal pentapeptide amide sequence motif (-FXGLM-amide) that also constitutes the bioactive core of the peptide. Amidation of the C-terminal methioninyl residue appears to be mandatory in the expression of biological activity. Here, we describe the isolation, characterization and molecular cloning of a novel tachykinin named ranachensinin, from the skin secretion of the Chinese brown frog, Rana chensinensis. This peptide, DDTSDRSN QFIGLM-amide, contains the classical C-terminal pentapeptide amide motif in its primary structure and an Ile (I) residue in the variable X position. A synthetic replicate of ranachensinin, synthesized by solid-phase Fmoc chemistry, was found to contract the smooth muscle of rat urinary bladder with an EC50 of 20.46 nM. However, in contrast, it was found to be of low potency in contraction of rat ileum smooth muscle with an EC50 of 2.98 µM. These data illustrate that amphibian skin secretions continue to provide novel bioactive peptides with selective effects on functional targets in mammalian tissues.

Analysis of epistasis in linked regions in the Irish study of high-density schizophrenia families

Epistasis may be important in the etiology of schizophrenia. Analysis of epistasis has been important in the positional cloning of a gene involved in the etiology of type II diabetes mellitus. We investigated the importance of epistasis among six linked regions in 268 multiplex pedigrees in the Irish Study of High-Density Schizophrenia Families (ISHDSF) by computing pairwise correlations between nonparametric linkage scores for narrow, intermediate, and broad diagnostic definitions. The linked regions were on chromosomes 2, 4, 5, 6, 8, and 10. No correlation reached our a priori level of statistical significance. Using this statistical approach, we did not find evidence of important epistatic effects among these six regions in the ISHDSF.

Macrofaunal assemblages from mud volcanoes in the Gulf of Cadiz

Desde a descoberta em 1999 do primeiro vulcão de lama no Golfo de Cádis, cerca de 40 locais, de profundidade variável entre os 200 e os 3900 m, com diferentes graus de emissão de hidrocarbonetos foram localizados e amostrados dentro do programa IOC-UNESCO “Training Through Research (TTR) “ e mais recentemente dentro do projecto europeu HERMES. Neste estudo investigamos as comunidades da macrofauna dos vulcões de lama do Golfo de Cádis utilizando uma diversidade de equipamento de amostragem quantitativo e não quantitativo. Mais de 14550 espécimes foram examinados e incluídos nos diferentes grupos taxonómicos, sendo fornecida uma lista taxonómica detalhada com o menor nível taxonómico possível. A biodiversidade, distribuição dos principais taxa, as espécies quimiossintéticas e a biodiversidade regional e substituição de espécies são apresentados e discutidos. Dentro da macrofauna, os bivalves (nomeadamente super-familia Thyasiroidea, espécies quimisimbióticos e comunidade de bivalves) e os ofiurideos são estudados em pormenor. Os Thyasiroidea colhidos nos vulcões de lama do Golfo de Cádis são revistos. Das sete espécies identificadas, apenas uma Thyasira vulcolutre. sp. nov se encontra associada a um ambiente quimiossintético. Esta espécie é restrita a locais activos, mas não se verificam padrões de distribuição para as outras espécies. Os bivalves quimiosimbióticos amostrados são revistos. Das 10 espécies fortemente associadas a ambientes quimiossintéticos duas Solemyidae, Petrasma elarraichensis sp. nov. e Acharax gadirae sp. nov., uma Lucinidae, Lucinoma asapheus sp. nov., e uma Vesicomyidae, Isorropodon megadesmus sp. nov. são descritas e comparadas com similares das respectivas famílias. As comunidades de bivalves foram analisadas em detalhe e do estudo de 759 espécimes (49 espécies em 21 familias) descreve-se a diversidade e padrões de distribuição. Os Ophiuroidea amostrados nos vulcões de lama e ambientes batiais adjacentes são revistos. Treze espécies são incluídas em 4 famílias, Ophiacanthidae, Ophiactidae, Amphiuridae e Ophiuridae e são identificadas, tendo sido descrita uma nova espécie Ophiopristis cadiza sp. nov. Rácios isotópicos (δ13C, δ15N, δ34S) foram determinados em várias espécies no intuito de investigar a ecologia trófica das comunidades bênticas dos vulcões do Golfo de Cádis. Os valores de δ13C para os bivalves Solemyidae, Lucinidae e Thyasiridae estão de acordo com os valores para outros bivalves conhecidos por possuírem simbiontes tiotróficos. Por outro lado os valores de δ13C e δ34S para Bathymodiolus mauritanicus sugerem a ocorrência de metanotrofia. A análise da fauna heterotrófica indica igualmente que as espécies habitantes da cratera dos vulcões de lama derivam a sua nutrição de fontes quimiossintéticas. A indicação pela análise isotópica que as bactérias autotróficas contribuem substancialmente para a nutrição dos bivalves hospedeiros, levou-nos a investigar os endossimbiontes e as suas relações filogenéticas relativamente a outros bivalves através da análise comparativa de análises de sequências de 16S ribossomal RNS. Análises moleculares PCR-DGGE (Denaturing Gradient Gel Electrophoresis) e clonagem de genes de bacterias 16S rRNA confirmaram a presença de simbiontes oxidantes de enxofre e colocam a possibilidade de uma simbiose dupla para o B. mauritanicus. A diversidade microbiana dentro dos Frenulata foi igualmente estudada recorrendo a métodos moleculares e revelou a não existência de padrão entre espécies, vulcões, profundidade e idade do animal sugerindo assim a não procura de simbiontes específicos.

The downy mildew resistance locus Pp523 is located on chromosome C8 of Brassica oleracea L.

We have previously constructed a genetic map of Brassica oleracea L. containing the Pp523 locus that confers downy mildew resistance to adult plants. In this work, 44 SSR markers of reference for the Brassica C genome chromosomes were added to the map, allowing the nine major linkage groups to be assigned to the nine chromosomes of B. oleracea. Locus Pp523 was located on chromosome C8, and a locus determining flower colour was mapped to chromosome C3. In comparison with the first version of the map, the new map is denser and more compact. The available genomic information on B. oleracea was enriched with the chromosome location of two phenotypic traits and 421 DNA markers (RAPD, ISSR, AFLP, SCAR, BAC-end derived STS, SSR and other PCR markers). Conversely, the genomic information on B. oleracea chromosome C8 is being used as an additional tool for the map-based cloning of Pp523, the first gene for adult plant resistance to downy mildew precisely located to a specific chromosome of this crop species.

The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.

Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.