Molecular cloning,characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp,Catla catla

The primordial immunoglobulin class, IgD, was the first non‐IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (Cc IgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of Cc IgD exhibited greatest similarity with Ctenopharyngodon idella . qRT‐PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post‐Aeromonas hydrophila challenge. Cc IgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in Cc IgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

     

Loss of genetic variation in hatchery-reared Indian major carp, Catla catla

The hypothesis that effective population sizes are low in hatchery-reared catla ( Catla catla ) from Bangladesh, possibly leading to inbreeding and loss of variation, was tested. The study was based on analysis of seven microsatellite loci in three samples of hatchery-reared catla and four samples representing wild populations. Pair-wise estimates of genetic differentiation between samples were low between wild samples (θ ranging from 0·012 to 0·034), but high between hatchery samples (θ ranging from 0·153 to 0·185), suggesting strong genetic drift in hatcheries. Genetic variation, both in terms of expected heterozygosity and allelic richness, was significantly lower in hatchery samples than in samples of wild catla. Application of a method for reconstructing families among offspring without parental genetic data showed that the hatchery samples consisted of very few half- and full-sib families, whereas the wild samples consisted of a high number of families, suggesting that most individuals were unrelated. Finally, estimation of the effective number of parents ( N _b) in the largest sample of hatchery fish confirmed that effective population size was low ( N _b= 14·9 for multiallelic loci and N _b= 10·6 if alleles were pooled into two composite alleles). The results show that low effective population sizes leading to loss of variation and possibly inbreeding depression should be a matter of serious concern in aquaculture production of catla.     

A new epithelial-like cell line from eye muscle of catla Catla catla (Hamilton): development and characterization

A new cell line [Sahul India Catla Eye (SICE)] has been developed from eye tissue of Indian major carp ( Catla catla ), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 15% foetal bovine serum (FBS). These cells have been subcultured >80 times over a period of 1·5 years. This cell line has been designated SICE. The SICE cell line consists predominantly of epithelial-like cells. These cells are strongly positive for epithelial markers such as pancytokeratin and cytokeratin 19. The cells were able to grow at temperatures between 25 and 32° C with optimum temperature of 28° C. The growth rate of catla eye cells increased as the FBS proportion increased from 2 to 20% at 28° C with optimum growth at the concentrations of 15 or 20% FBS. Six marine fish viruses (fish nervous necrosis virus, marine birnavirus-NC1, chum salmon virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus-Sp and hirame rhabdovirus) were tested on this cell line to determine its susceptibility. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the fourth day after subculture. Polymerase chain reaction amplification of mitochondrial 12S rRNA indicated identity of these cell lines with those reported from this animal species, confirming that the cell lines were of catla origin. The cells were successfully cryopreserved and revived at passage numbers 10, 25, 40 and 60. The cell cycle analysis by fluorescence-activated cell sorter revealed that most of the cells on the second day of culture were in S-phase, indicating a high growth rate. When the SICE cells were transfected with pEGFP vector DNA, significant fluorescent signals were observed suggesting that the SICE cell line can be a useful tool for transgenic and genetic manipulation studies.     

印度北方河流中主要鲤科鱼类卡拉(鱾)的年龄和生长

为了获得印度北部赣达(Ganga)盆地河流中野生卡特拉(鱾)种群的年龄结构和重要生长参数,对该鱼的年龄和生长进行了研究.鳞片取自商业捕捞和实验室饲养的样品.根据研究分析,该鱼最大年龄可达8龄;巴吉拉蒂河(Bhagirathi R.)的种群平均体长为521.51 mm,退算体长为288.9-1132.3 mm;旁遮普邦(Punjab)Satluj河种群平均体长为641.6 mm,退算体长为335.4-1096.08 mm.2龄时,种群线性生长率(Cl)和体重增加率(Cw)表现出迅速下降的趋势.其它生长参数值(Clt)也呈现快速下降.退算体长差异(ANOVA)分析显示,生活在赣达盆地不同流域中的种群,1 -4 龄组的长度差异较明显(P<0.05),高龄组(5 -8 )差异不显著.根据本项研究结果,提出了对印度北部赣达盆地相关河流中生活的野生卡特拉(鱾)种群资源持续利用的对策.     

Identification,cDNA Cloning,and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla

Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhβ deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhβ protein comprises of five β-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.     

Computer‐aided assessment of the chemokine receptors CXCR3, CXCR4 and CXCR7 expression in gallbladder carcinoma

Gallbladder carcinoma (GBC) is a vicious and invasive disease. The major challenge in the clinical treatment of GBC is the lack of a suitable prognosis method. Chemokine receptors such as CXCR3, CXCR4 and CXCR7 play vital roles in the process of tumour progression and metastasis. Their expression levels and distribution are proven to be indicative of the progression of GBC, but are hard to be decoded by conventional pathological methods, and therefore, not commonly used in the prognosis of GBC. In this study, we developed a computer‐aided image analysis method, which we used to quantitatively measure the expression levels of CXCR3, CXCR4 and CXCR7 in the nuclei and cytoplasm of glandular and interstitial cells from a cohort of 55 GBC patients. We found that CXCR3, CXCR4 and CXCR7 expressions are associated with the clinicopathological variables of GBC. Cytoplasmic CXCR3, nuclear CXCR7 and cytoplasmic CXCR7 were significant predictive factors of histology invasion, whereas cytoplasmic CXCR4 and nuclear CXCR4 were significantly correlated with T and N stage and were associated with the overall survival and disease‐free survival. These results suggest that the quantification and localisation of CXCR3, CXCR4 and CXCR7 expressions in different cell types should be considered using computer‐aided assessment to improve the accuracy of prognosis in GBC.     

Molecular cloning,structural analysis,and expression of carp ZP2 gene

The cDNAs encoding carp ZP2 homologous to winter flounder and mammalian ZP2 were cloned. Carp ZP2 contains a tandemly repetitive domain and a nonrepetitive domain. A repeat is composed of 13 amino-acid residues whose consensus sequence is QQTSQQFQPQKPA/V. The length of the repetitive domain is highly variable, but that of the nonrepetitive domain is fairly constant among various cDNAs. The termination codons of various cDNAs appear at three different positions. Three groups of cDNAs were therefore categorized. Groups I–III encode a nonrepetitive domain of 356, 255, and 10 residues, respectively. A carp ZP2 gene corresponding to group II cDNA was cloned. It spans 2.4 kb and consists of eight exons and seven introns. Carp ZP2 mRNA was detected only in oocytes but not in other tissues. Carp ZP2 is heterogenous in size. The molecular weight ranges from 40–80 kDa. It is present in vitellogenic but not in previtellogenic oocytes, nor in other tissues. Carp ZP2 content in oocytes increases as vitellogenesis proceeds. Mol. Reprod. Dev. 46:258–267, 1997. © 1997 Wiley-Liss, Inc.     

大鼠CXCR4基因RNAi慢病毒载体的构建及其在骨髓间质干细胞中的表达

为深入研究CXCR4在骨髓间质干细胞(MSCs)体内迁移中的作用, 构建CXCR4基因RNA干扰(RNAi)慢病毒载体并实现其在大鼠MSCs (rMSCs)中表达。根据大鼠CXCR4 mRNA序列, 设计并合成包含各靶序列的互补DNA链,插入pSUPER载体的H1 RNA启动子后面, 产生pRiCXCR4, 将其中的CXCR4 shRNA表达结构酶切插入慢病毒载体质粒pNL-EGFP, 产生pNL-RiCXCR4-EGFP。在脂质体介导下与包装质粒pHELPER和包膜质粒pVSVG共转染293T细胞, 包装生产慢病毒,测定慢病毒功能滴度。慢病毒转导rMSCs后, 用Real-time RT-PCR、Western blotting和流式细胞术检测RNAi组(CXCR4a、CXCR4b和CXCR4c)、空载体组(Mock)和对照组(Control)中CXCR4表达情况。结果显示, 酶切和测序证实pRiCXCR4质粒构建正确, 产生能同时表达增强型绿色荧光蛋白(EGFP)和CXCR4 shRNA的慢病毒载体质粒pNL-RiCXCR4-EGFP, 未浓缩和浓缩慢病毒悬液的功能滴度分别为6.4×104TU/mL和6.9×106TU/mL。慢病毒转导rMSCs 48 h后, 与空载体组和空白组相比, 3个RNAi组均不同程度抑制CXCR4表达, CXCR4b-MSC组在mRNA水平抑制了95.6%, 抑制作用最明显。大鼠CXCR4基因RNAi慢病毒载体构建成功, 为深入研究CXCR4在rMSCs向损伤组织定向迁移的作用奠定了基础。     

Genetic variation of wild and hatchery populations of the catla Indian major carp (Catla catla Hamilton 1822: Cypriniformes, Cyprinidae) revealed by RAPD markers

Genetic variation is a key component for improving a stock through selective breeding programs. Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic variation in three wild population of the catla carp (Catla catla Hamilton 1822) in the Halda, Jamuna and Padma rivers and one hatchery population in Bangladesh. Five decamer random primers were used to amplify RAPD markers from 30 fish from each population. Thirty of the 55 scorable bands were polymorphic, indicating some degree of genetic variation in all the populations. The proportion of polymorphic loci and gene diversity values reflected a relatively higher level of genetic variation in the Halda population. Sixteen of the 30 polymorphic loci showed a significant (p < 0.05, p < 0.01, p < 0.001) departure from homogeneity and the F(ST) values in the different populations indicated some degree of genetic differentiation in the population pairs. Estimated genetic distances between populations were directly correlated with geographical distances. The unweighted pair group method with averages (UPGMA) dendrogram showed two clusters, the Halda population forming one cluster and the other populations the second cluster. Genetic variation of C. catla is a useful trait for developing a good management strategy for maintaining genetic quality of the species.     

Glycosylated molecular variants of C-reactive proteins from the major carp Catla catla in fresh and polluted aquatic environments

Elevated level of pollutant specific glycosylated molecular variants of C-reactive protein have been purified to electrophoretic homogeneity from the sera of major carp, Catla catla confined in freshwater (CRP_N) and water polluted with nonlethal doses of cadmium (CRP_Cd), mercury (CRP_Hg), phenol (CRP_Ph) and hexachlorocyclohexane (CRP_Hex). These CRPs differ amongst themselves in electrophoretic mobility, and in their carbohydrate content ranging from 20–50%. CRPs interact with pneumococcal C-polysaccharide (CPS) showing different binding constants. Both phosphorylcholine (PC) and calcium are indispensable for binding. Studies on amino acid compositions, electrophoretic analysis, isoelectric focusing, binding to PC & CPS and secondary structures of the purified CRPs indicate, that, they differ from each other. However, they share the common properties of a CRP, including pentraxin structure revealed by electron microscopy. Taken together, our results provide a new structural insight regarding the connection between the presence of unique molecular variants and probably the toxicity therein combated.     

趋化因子及趋化因子受体3在自身免疫疾病中的作用

人体在防御和清除入侵病原体等异物时,有一种使白细胞趋集的功能,有一些低分子量的物质能引起这种功能称之为趋化剂或趋化因子。这些小蛋白因其有定向细胞趋化作用而得名。经研究表明,趋化因子受体3（CXCR3）趋化因子可能在自身免疫内分泌疾病中起到致病作用。此外,血清中CXCR3趋化因子的判定可能辅助检测免疫活性。CXCR3和优先参与趋化Th1细胞的因子。该受体连接的趋化因子10（CXCL10）不仅参与白细胞募集,还诱导T细胞增殖的异源体和抗原的刺激。趋化因子10正调节Th1细胞产物并且负调节Th2细胞的产物。免疫反应纤维结合素（INF）产物可增强特异的炎症反应。当被激活或者发现炎症和肿瘤细胞后趋化因子受体3-B在内皮细胞中表达并且其结合的趋化因子10,趋化因子9和趋化因子11激活后产生血管抑制作用。     

Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

Design,synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]_n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]_n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL12_1–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC₅₀ values of <50 nM and <800 nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG₃]₂ linked homodimeric DV3, was an effective CXCR4 antagonist (IC₅₀ = 22 nM); and ligand 21, a [PEG₃]₂ linked heterodimeric DV3–CXCL12_1–8, was an effective CXCR4 agonist (IC₅₀ = 407 nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.     

CXCR3, a double-edged sword in tumor progression and angiogenesis

CXC chemokines are involved in chemotaxis, regulation of cell growth, induction of apoptosis and modulation of angiostatic effects. CXCL9, CXCL10, CXCL11, CXCL4 and its variant CXCL4L1 are members of the CXC chemokine family, which bind to the CXCR3 receptor to exert their biological effects. These chemokines are associated with a variety of human diseases including chronic inflammation, immune dysfunction, cancer and metastasis. In this review, we focus on accumulating evidence demonstrating the pivotal role of CXCR3 in tumor progression. Its effects are mediated directly in tumor cells or indirectly through the regulation of angiogenesis and tumor immunity. Understanding the emerging role of CXCR3 and its signaling mechanisms further validates this receptor as a biomarker and therapeutic target for tumor progression and tumor angiogenesis.     

Molecular cloning, characterization and expression analysis of thrombospondin gene from Penaeus monodon

In present study, a thrombospondin gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length P. monodon thrombospondin (PmTSP) cDNA contained a 5' untranslated region (UTR) of 9 bp, an open reading frame (ORF) of 2778 bp encoding a polypeptide of 925 amino acids with molecular mass 100.57 kDa, and a 3'UTR of 99 bp. ScanProsite analysis indicated that PmTSP contained four chitin-binding type-II domains, an EGF-like domain, eight thrombospondin type-III repeats and one thrombospondin C-terminal domain. Homology analysis of the deduced amino acid sequence of the PmTSP with other known TSP sequences by MatGAT software revealed that the PmTSP shows very high homology with the sequences of Fennerpenaeus chinensis (89.9% similarity, 83.8% identity). Analysis of the tissue expression pattern of the PmTSP gene showed that the PmTSP mRNA was expressed in all tested tissues, including hepatopancreas, ovary, muscle, intestine, neurosecretory organ in eyestalk, neurosecretory organ in brain, stomach, and heart, with highest level in the ovary. Furthermore, the PmTSP expression was found to be of high level in six development stages of the ovary. The results indicated that PmTSP might play an important role in ovarian development.     

人趋化因子受体CXCR4基因的克隆及原核表达

目的:构建人CXCR4原核表达载体并在大肠杆菌中进行表达。方法:从健康人外周血单个核细胞提取总RNA,以RT-PCR获得CXCR4基因全长1059bp的完整编码序列,将其克隆入载体pMD-18T中,经限制性内切酶和菌落PCR分析并测序证实的阳性重组子与表达载体pET-28a( )连接并转化大肠杆菌BL21(DE3)。结果:12%SDS-PAGE分析表明,在30℃以IPTG诱导获得分子量为43ku的His-CXCR4融合蛋白表达带,诱导4h后此蛋白表达量约为全菌总蛋白的25%。结论:成功获得人CXCR4基因融合蛋白。