Genotyping of single nucleotide polymorphisms in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Decapoda,Penaeidae) by tetra-primer ARMA-PCR

This study introduced a kind of new SNP genotyping strategy in genetic analysis of Chinese shrimp (Fenneropenaeus chinensis). Twenty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of six selected families of marine shrimp F. chinensis. The effective number of alleles ranged from 1.127 to 1.993, with an average value of 1.600. The average values of expected and observed heterozygosities of the SNPs ranged from 0.505∼0.609 and 0.373∼0.487, respectively. Polymorphism information content (PIC) values ranged from 0.145 to 0.373. Among 120 population-locus cases (six populations × twenty loci), only 8 (6.7%) showed significant deviation (P < 0.05), while the other 112 (93.3%) were in Hardy-Weinberg equilibrium (HWE) (P > 0.05). The frequencies of minor alleles ranged from 0.378 to 0.497. For the six families, the wild genotype and its allele frequency were significantly higher than that of the mutant genotype and its allele frequency. Fifty five point forty two percent of the population-locus showed heterozygosity. The results indicated that tetra-primer ARMA-PCR is a simple, rapid and efficient method for SNP genotyping which make it useful in a broad aspects of F. chinensis genetic and breeding studies.     

Adaptive haemoglobin gene control in Daphnia pulex at different oxygen and temperature conditions

Hypoxia-induced haemoglobin (Hb) expression is a central regulatory mechanism in Daphnia in response to environmental hypoxia or warm temperatures. Changes in Hb concentration as well as Hb subunit composition, which modulate Hb oxygen affinity, guarantee the oxygen supply of tissues under these environmental conditions. Based on the sequenced D. pulex genome, Hb genes were related to the properties of haemolymph Hb, which included its concentration and oxygen affinity (both measured by spectrophotometry) as well as the Hb subunit composition (determined by 2-D gel electrophoresis and ESI-MS analysis). Permanent cultures of D. pulex acclimated to different oxygen conditions (normoxia and hypoxia) and temperatures (10°C, 20°C, and 24°C), showed characteristic changes in Hb concentration, subunit composition and oxygen affinity. Several subunits (Hb4, Hb7, Hb8, and Hb10) were obviously responsible for changes in oxygen affinity including those, which carry a number of hypoxia-responsive elements (HREs) upstream of the respective gene (hb4 and hb10). Analysing the effects of different oxygen- or temperature-acclimations on Hb subunit expression in D. pulex and D. magna on a common basis (Hb concentration or oxygen affinity) revealed a general pattern of oxygen and temperature effects on Hb, which implies that Hb quantity and quality are mostly influenced by the degree of tissue hypoxia. Differences between both species in the onset of hypoxia-induced differential Hb expression and Hb oxygen affinity, which are probably related to different HRE patterns and functionally important differences in the amino acid sequence of only a few subunits, cause a reduced ability of D. pulex to adjust Hb function to temperature changes in comparison to D. magna.     

Engineering biological systems with synthetic RNA molecules

RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors.     

CYP-eicosanoids--a new link between omega-3 fatty acids and cardiac disease?

Fish oil omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against arrhythmia and sudden cardiac death by largely unknown mechanisms. Recent in vitro and in vivo studies demonstrate that arachidonic acid (AA) metabolizing cytochrome P450-(CYP) enzymes accept EPA and DHA as efficient alternative substrates. Dietary EPA/DHA supplementation causes a profound shift of the cardiac CYP-eicosanoid profile from AA- to EPA- and DHA-derived epoxy- and hydroxy-metabolites. CYP2J2 and other CYP epoxygenases preferentially epoxidize the ω-3 double bond of EPA and DHA. The corresponding metabolites, 17,18-epoxy-EPA and 19,20-epoxy-DHA, dominate the CYP-eicosanoid profile of the rat heart after EPA/DHA supplementation. The (ω-3)-epoxyeicosanoids show highly potent antiarrhythmic properties in neonatal cardiomyocytes, suggesting that these metabolites may specifically contribute to the cardioprotective effects of omega-3 fatty acids. This hypothesis is discussed in the context of recent findings that revealed CYP-eicosanoid mediated mechanisms in cardiac ischemia-reperfusion injury and maladaptive cardiac hypertrophy.     

Interaction of Acinetobacter baumannii 19606 and 1656-2 with Acanthamoeba castellanii

Acinetobacter baumannii is virtually avirulent for healthy people but maintains a high virulence among critically ill patients or immuno-compromised individuals. The ability of A. baumannii to adhere to cells and persist on surfaces as biofilms could be central to its pathogenicity. In the present study, we compared the virulence of the A. baumannii 1656-2 clinical strain, which is able to form a thick biofilm, with the virulence of the A. baumannii type strain (ATCC 19606^T). Acanthamoeba castellanii, a single-celled organism, was used as the host model system to study the virulence of A. baumannii. Compared to A. baumannii ATCC 19606^T, A. baumannii 1656-2 exhibited a higher ability to adhere and invade A. castellanii cells and had a higher killing rate of A. castellanii cells. Furthermore, co-incubation of the amoeba cells and the cell-free supernatant of A. baumannii resulted in the cell death of the amoebae. Heat inactivation or proteinase K treatment of the supernatant did not eliminate its cytotoxicity, suggesting heat stable non-protein factors are responsible for its cytotoxicity to A. castellanii cells. In conclusion, this study for the first time has revealed the capacity of the A. baumannii strain and/or its metabolic products to induce cytotoxicity in A. castellanii cells.     

A defensin‐like antimicrobial peptide from the venoms of spider,Ornithoctonus hainana

The defensin‐like antimicrobial peptides have been characterized from various other arthropods including insects, scorpions, and ticks. But no natural spider defensin‐like antimicrobial peptides have ever been isolated from spiders, except couple of cDNA and DNA sequences of five spider species revealed by previous genomic study. In this work, a defensin‐like antimicrobial peptide named Oh‐defensin was purified and characterized from the venoms of the spider, Ornithoctonus hainana. Oh‐defensin is composed of 52 amino acid (aa) residues including six Cys residues that possibly form three disulfide bridges. Its aa sequence is MLCKLSMFGAVLGV PACAIDCLPMGKTGGSCEGGVCGCRKLTFKILWDKKFG. By BLAST search, Oh‐defensin showed significant sequence similarity to other arthropod antimicrobial peptides of the defensin family. Oh‐defensin exerted potent antimicrobial activities against tested microorganisms including Gram‐positive bacteria, Gram‐negative bacteria, and fungi. The cDNA encoding Oh‐defensin precursor was also cloned from the cDNA library of O. hainana. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris

The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunologic research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant.