The modified Peierls-Nabarro model of interfacial misfit dislocation

The Peierls-Nabarro model of the interfacial misfit dislocation array is analytically extended to a family of dislocations of greater widths. By adjusting a parameter, the width of the misfit dislocations, the distribution of the shear stress, and the restoring force law can be systematically varied. The smaller the amplitude of the restoring force, the wider the misfit dislocations and the lower the interfacial energy.

Multiple bombesin-like peptides with opposite functions from skin of Odorrana grahami

Bombesin-like peptides (BLPs) are a family of neuroendocrinic peptides that mediate a variety of biological activities. Three mature BLPs from the skin secretions of the frog Odorrana grahami were purified. Several bombesin-like peptide cDNA sequences enc

A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms. (C) 2008 Elsevier Inc. All rights reserved.

Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-gamma production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog, Rana nigrovittata

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents. (C) 2009 Elsevier Inc. All rights reserved

Five novel antimicrobial peptides from skin secretions of the frog, Amolops loloensis

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb): the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana. (C) 2009 Elsevier Inc. All rights reserved.

Phylogenetic analyses of Fringillidae (Aves : Passeriformes) using mitochondrial tRNA gene sequences and secondary structure

Fringillidae is a large and diverse family of Passeriformes. So far, however, Fringillidae relationships deduced from morphological features and by a number of molecular approaches have remained unproven. Recently, much attention has been attracted to mitochondrial tRNA genes, whose sequence and secondary structural characteristics have shown to be useful for Acrodont Lizards and deep-branch phylogenetic studies. In order to identify useful phylogenetic markers and test Fringillidae relationships, we have sequenced three major clusters of mitochondrial tRNA genes from 15 Fringillidae, taxa. A coincident tree, with coturnix as outgroup, was obtained through Maximum-likelihood method using combined dataset of 11 mitochondrial tRNA gene sequences. The result was similar to that through Neighbor-joining but different from Maximum-parsimony methods. Phylogenetic trees constructed with stem-region sequences of 11 genes had many different topologies and lower confidence than with total sequences. On the other hand, some secondary structural characteristics may provide phylogenetic information on relatively short internal branches at under-genus level. In summary, our data indicate that mitochondrial tRNA genes can achieve high confidence on Fringillidae phylogeny at subfamily level, and stem-region sequences may be suitable only at above-family level. Secondary structural characteristics may also be useful to resolve phylogenetic relationship between different genera of Fringillidae with good performance.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C-20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants. temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (>= 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH similar to 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues. (C) 2009 Elsevier B.V. All rights reserved.

Kinetic study of the 2-methyl-3-methoxy-4-phenylbutanoic acid produced by oxidation of microcystin in aqueous solutions

Microcystins (MCs) are a family of related cyclic hepatotoxic heptapeptides, of which more than 70 types have been identified. The chemically unique nature of the C20 beta-amino acid, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca4,6-dienoic acid (Adda), portion of the MCs has been exploited to develop a strategy to analyze the entirety. Oxidation of MCs causes the cleavage of MC Adda to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB). In the present study, we investigated the kinetics of MMPB produced by oxidation of the most-often-studied MC variant, MC-LR (L = leucine, R = arginine), with permanganate-periodate. This investigation allowed insight regarding the influence of the reaction conditions (concentration of the reactants, temperature, and pH) on the conversion rate. The results indicated that the reaction was second order overall and first order with respect to both permanganate and MC-LR. The second-order rate constant ranged from 0.66 to 1.35 M/s at temperatures from 10 to 30 degrees C, and the activation energy was 24.44 kJ/mol. The rates of MMPB production can be accelerated through increasing reaction temperature and oxidant concentration, and sufficient periodate is necessary for the formation of MMPB. The initial reaction rate under alkaline and neutral conditions is higher than that under acidic conditions, but the former decreases faster than the latter except under weakly acidic conditions. These results provided new insight concerning selection of the permanganate-periodate concentration, pH, and temperature needed for the oxidation of MCs with a high and stable yield of MMPB.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA Library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full Length cDNA of carp SLP-76 was 2007 bp, consisting of a T-terminal untranslated region (UTR) of 285 bp, a T-terminal. UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homotogues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts. (c) 2007 Published by Elsevier Ltd.

Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

Hir/Hira (histone regulation) genes were first identified in yeast as negative regulators of histone gene expression. It has been confirmed that HIRA is a conserved family of proteins present in various animals and plants. In this paper, the cDNAs of the Hira homolog named CagHira and CaHira were isolated from gynogenetic gibel carp (gyno-carp) and gonochoristic color crucian carp (gono-carp) respectively. The full-length CagHira is 3,860 bp in length with an open reading frame (ORF) of 3,033 bp that encodes 1,011 amino acids, while the full-length CaHira is 3,748 bp in length and also has an ORF of 3,033 bp. The deduced amino acid sequences of both Hira homologs contain seven WD domains and show high identity with other HIRA family members. RT-PCR analyses revealed strong expression of Hira in the ovaries, whereas no expression was detected in the testes of either of the fishes. Hira transcription was not detected in the liver of gyno-carp, but a high level of Hira mRNA was observed in gono-carp. The temporal expression pattern showed that the Hira mRNA is consistently expressed during all embryonic development stages in gyno-carp. However, the abundance of CaHira mRNA significantly decreased (P < 0.05) shortly after fertilization and then increased again and remained stable from gastrula till hatching. The varying spatiotemporal expression patterns of Hira genes in gyno-carp and gono-carp may be associated with the differing reproductive modes used by these two closely related fishes. Our results suggest that Hira may play a role not only in the decondensation of sperm nucleus and the formation of pronucleus during fertilization, but also in gastrulation and the subsequent development of embryos.

Identification of the Angel-related elements in cyprinid fishes and their phylogenetic implications

Angel-related element belongs to the family of miniature inverted-repeat transposable elements (MITEs). In this paper we report the identification of an Angel-related element in the series Leuciscini of cyprinid fishes, which is located in the second intron of the growth hormone (GH) gene. We have also found that this element is absent in orthologous locus in the series Barbini of cyprinid fishes, that provides new evidence for the monophyly of the series Leuciscini. The insertion of Angel-related element into the GH gene might take place in the common ancestor of the series Leuciscini after its divergence from the series Barbini. The high sequence divergence and relatively broad species distribution of Angel-related elements implies that they might be ancient transposons which appeared about 26 million years ago.

Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperons and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type 11 Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp4OA4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells. (c) 2005 Elsevier Ltd. All rights reserved.

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6 - 99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G(4)

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.