Compatibilization effect of graft copolymer on immiscible polymer blends: 1. LLDPE/SBS/LLDPE-g-PS systems

compatibilizing effect of graft copolymer, linear low density polyethylene-g-polystyrene (LLDPE-g-PS), on immiscible blends of LLDPE with styrene-butadiene-styrene triblock copolymer (SBS) has been investigated by means of C-13 CPMAS n.m.r. and d.s.c. techniques. The results indicate that LLDPE-g-PS is an effective compatibilizer for LLDPE/SBS blends. It was found that LLDPE-g-PS chains connect two immiscible components, LLDPE and SBS, through solubilization of chemically identical segments of LLDPE-g-PS into the amorphous region of LLDPE acid PS block domain of SBS, respectively. It was also found that LLDPE-g-PS chains connect the crystalline region of LLDPE by isomorphism, with serious effects on the supermolecular structure of LLDPE. The effect of LLDPE-g-PS on the supermolecular structure of LLDPE in the LLDPE/SBS blends obviously depends on the composition of the blends, but has little dependence on the PS grafting yields of LLDPE-g-PS. (C) 1998 Elsevier Science Ltd. All rights reserved.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares similar to 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia colt, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular beta-agarase. E. coli DH5 alpha harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5 alpha/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5 alpha/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5 alpha/pTAET21, is an effective vaccine candidate against E. tarda infection. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a T-terminal untranslated region (UTR) of 62 bp and a 3' UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6 h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21 (DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LIDS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk. (c) 2008 Elsevier Ltd. All rights reserved.

Cloning and recombinant expression of a crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis

Antimicrobial peptides or proteins (AMPs) are proved to be one of the most important humoral factors to resist pathogen infection. As an antimicrobial protein, crustin had been described in invertebrates as a component of the innate immune system. A crustin-like gene (CruFc) was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5'-RACE PCR. The full-length cDNA consists of 523 with 405 bp open reading frame encoding 134 amino acids and the deduced peptide contains a putative signal peptide of 17 amino acids. The sequence also contains a whey-acidic protein (WAP) domain at the C-terminal. Transcripts of CruFc were mainly detected in haemocytes and gill by RT-PCR analysis. In addition, another full-length cDNA named CshFc was also cloned from haemocytes of Chinese shrimp and its inferred amino acid sequence lacks the WAP-type 'four-disulfide core' domain. The fusion proteins containing CruFc and CshFc were, respectively, produced and the antimicrobial assays revealed that the recombinant CruFc could inhibit the growth of grain-positive bacteria in vitro but the recombinant CshFc could not inhibit at the same conditions. The difference of antimicrobial activity between recombinant CruFc and CshFc provides the evidence that the four-disulfide core domain of crustin may play an important role in its biological function. (c) 2006 Elsevier B.V. All rights reserved.

Broader pattern of tandem repeats in the mitochondrial control region of Perciformes

Perciformes, the largest order of vertebrates with 20 suborders, is the most diverse fish order that dominates vertebrate ocean life. The complete mitochondrial control region (CR) of Trichiurus japonicus (Trichiuridae, Scombroidei) and Pampus sp. (Stromateidae, Stromateoidei) were amplified and sequenced. Together with data from GenBank, the tandem repeats in the mitochondrial CR from 48 species, which covered nine suborders of Perciformes, are reported in this study. The tandem repeats tend to be long in the suborder Percoidei and Stromateoidei. The identical repeats in 21 species of Cichlidae suggest a common origin and have existed before species divergence. Larimichthys crocea shows tandem repeats instead of the typical structure of the central conserved sequence blocks, which was first reported in Perciformes and vertebrates. This might have resulted from interruption of the polymerase activity during the H-strand synthesis. The four broader patterns presented here for the tandem repeats, including those in both the 5' and 3' ends, only in the either 5' or 3' end, and in the central conserved domain of the control region, will be useful for understanding the evolution of species.

Differential regulation of Sciaenops ocellatus viperin expression by intracellular and extracellular bacterial pathogens

Viperin is an antiviral protein that has been found to exist in diverse vertebrate organisms and is involved in innate immunity against the infection of a wide range of viruses. However, it is largely unclear as to the potential role played by viperin in bacterial infection. In this study, we identified the red drum Sciaenops ocellatus viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 5'-untranslated region (UTR) of 34 bp and a 3'-UTR of 350 bp. The deduced amino acid sequence of SoVip shares extensive identities with the viperins of several fish species and possesses the conserved domain of the radical S-adenosylmethionine superfamily proteins. Expressional analysis showed that constitutive expression of SoVip was relatively high in blood, muscle, brain, spleen, and liver, and low in kidney, gill, and heart. Experimental challenges with poly(I:C) and bacterial pathogens indicated that SoVip expression in liver was significantly upregulated by poly(I:C) and the fish pathogen Edwardsiella tarda but down-regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae. Similar differential induction patterns were also observed at cellular level with primary hepatocytes challenged with E. tarda, L anguillarum, and S. iniae. Infection study showed that all three bacterial pathogens could attach to cultured primary hepatocytes but only E. tarda was able to invade into and survive in hepatocytes. Together these results indicate that SoVip is involved in host immune response during bacterial infection and is differentially regulated at transcription level by different bacterial pathogens. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd tauri and Pd strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminat domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species. (C) 2007 Elsevier Ltd. All rights reserved.