Avaliação do efeito do inibidor de histona deacetilase Panobinostat (LBH589) no metabolismo energético de células derivadas de câncer de pulmão

Os mecanismos de ação citotóxica do inibidor de histona deacetilase LBH589 foram investigados em associação com o quimioterápico cisplatina (CDDP) em duas linhagens derivadas de câncer de pulmão de não pequenas células (CPNPC). Os resultados foram analisados em relação ao tipo de morte celular associada às alterações em enzimas relacionadas ao metabolismo energético e a via glicolítica. Para realização do trabalho, foram utilizadas as linhagens tumorais A549 (selvagem para o gene de p53) e Calu-1 (nulo para o gene de p53) tratadas com LBH589 em combinação ou não com CDDP. Foram realizadas curvas de tempo e dose-resposta com as drogas isoladamente pelo ensaio de viabilidade celular (MTT) nas duas linhagens para a escolha das melhores condições para o nosso estudo. As condições dos tratamentos isolados com redução da viabilida celular menores que o IC50 de cada fármaco foram selecionados para realização dos tratamentos combinados. As avaliações de apoptose foram realizadas por citometria de fluxo pelo ensaio de Anexina V/PI, e com a marcação de proteínas por Western Blotting. As proteínas relacionadas a via glicolítica foram avaliadas por Western Blotting e a expressão de RNAm por qPCR. Os resultados demonstraram que o LBH589 combinado a CDDP foi capaz de induzir apoptose em 70% das células (Calu-1) e 54,9% (A549) no tempo de 24 horas, e 90% (calu-1) e 62,1% (A549) em 48 horas, independendo, portanto, do status da p53. Os níveis de expressão de enzimas relacionadas com o metabolismos energético também sofreram alterações nos tratamentos estudados. O LBH589 induziu aumento de cerca de 4x dos níveis de RNAm de HK isoformas I e II em ambas as linhagens. Houve também um aumento na expressão proteica das isoformas de HK I e II. Outras enzimas relacionadas a via glicolítica como PFKP, PKM2 e LDHA foram analisadas e apresentaram redução da expressão proteica, principalmente na presença do LBH589. A combinação da CDDP com LBH589 parece ser promissora para o tratamento de CPNPC induzindo apoptose através de alterações no metabolismo energético tumoral.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS14 sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS14 at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development. (C) 2009 Elsevier Inc. All rights reserved.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins

Microcystins (MCs) are a potent liver tumor promoter, possessing potent tumor-promoting activity and weak initiating activity. Proto-oncogenes are known to be involved in the tumor-promoting mechanisms of microcystin-LR. However, few data are available on the effects of MCs oil proto-oncogenes in the whole animal. To investigate the effects of MCs on the expression profile of the proto-oncogenes in different organs, male Wistar rats were injected intravenously with microcystin extracts at a dose of 86.7 mu g MC-LR eq/kg bw (MC-LR eq, MC-LR equivalents). mRNA levels of three proto-oncogenes c-fos, c-jun and c-myc in liver, kidney and testis were analyzed using quantitative real-time PCR at several time points post-injection. Significant induction of these genes at transcriptional level was observed in the three organs. In addition, the increase of mRNA expression of all three genes was much higher in liver than in kidney and testis. Meanwhile, the protein levels of c-Fos and c-Jun were investigated by western blotting. Both proteins were induced in the three organs. However, elevations of protein levels were Much lower than those of mRNA levels. These findings suggest that the expression of c-fos, c-jun and c-myc might be one possible mechanism for the tumor-promoting activity and initiating activity of microcystins. (c) 2008 Published by Elsevier Ltd.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis

A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (I h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24 h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. (c) 2006 Elsevier GmbH. All rights reserved.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

Molecular cloning and characterisation of prophenoloxidase (ProPO) cDNA from Fenneropenaeus chinensis and its transcription injected by Vibrio anguillarum

The prophenoloxidase(ProPO) gene was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of prophenoloxidase gene consists of 3040 bp with a 2061 bp Open Reading Frame (ORF), encoding 686 amino acids. Phylogenetic analysis revealed that it belongs to insect-type invertebrate prophenoloxidase gene family. To understand ProPO reaction for pathogeny's challenge in shrimp, the expressions of ProPO in different tissues were studied by real-time PCR after challenged by Vibrio anguillarum. The results showed that the expression level of ProPO gene in haemocytes was highest among three studied tissues including haemocytes, lymphoid organ and hepatopancreas. The time-course change of ProPO mRNA levels in challenge experiment showed that ProPO mRNA transcripts had the biggest change extent in lymphoid organ.

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

Molecular characteristics and expression analysis of calreticulin in Chinese shrimp Fenneropenaeus chinensis

Calreticulin (CRT), as an endoplasmic reticulum luminal resident protein, plays important roles in Ca2+ homeostasis and molecular chaperoning. CRT on the surface of the cell can modulate cell adhesion, phagocytosis and integrin-dependent Ca2+ signaling. The full length cDNA of calreticulin (FcCRT) was cloned from Chinese shrimp Fenneropenaeus chinensis. It consists of 1672 by with an open reading frame of 1221 bp, encoding 406 amino acids. This is the first reported cDNA sequence of calreticulin in Crustacea. The deduced amino acid sequence of FcCRT showed high identity with those of Bombyx mori (88%), Drosophila melanogaster (83%), Mus musculus (82%) and Homo sapiens (82%). Highest expression of FcCRT was detected in ovary by Northern blot and in situ hybridization. Different mRNA levels of FcCRT were detected at various molting stages. Expression of FcCRT was induced significantly after 3 h of heat shock treatment, reached the maximum at 4 h and dropped after that. Differential expression profiles of FcCRT were observed in hepatopancreas and haemocytes when shrimp were challenged by white spot syndrome virus (WSSV). From the above results, we inferred that FcCRT might play important roles in Ca2+ homeostasis, chaperoning and immune function in shrimp. (c) 2007 Elsevier Inc. All rights reserved.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。