A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Pseudomonas duriflava sp nov., isolated from a desert soil

A Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain HR2(T) was isolated from a soil sample from the Talklimaken Desert in Xinjiang Province, China. Strain HR2(T) grew optimally at pH 7.0-8.0 and 30-37 degrees C in the presence of 0-1% (w/v) NaCl. An analysis of 16S rRNA gene sequences revealed that strain HR2(T) fell within the radiation of the genus Pseudomonas, the highest level of similarity being found with respect to Pseudomonas luteola IAM 13000(T) (97.5%); the levels of sequence similarity with respect to other recognized Pseudomonas species were < 96.4%. DNA-DNA hybridization showed that the genetic relatedness between strain HR2(T) and P. luteola IAM 13000(T) was 53.2%. The G + C content of the genomic DNA of strain HR2(T) was 55.2 mol%. The major fatty acids were 18: 1, summed feature 3 and 16:0. The hydroxylated fatty acids 10:0 3-OH, 12:0 3-OH and 12:0 2-OH were also present. The data obtained in this polyphasic study indicated that this isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas duriflava sp. nov. is proposed, The type strain is HR2(T) (=KCTC 221129(T) =CGMCC 1.6858(T)).

Sphingobacterium siyangense sp nov., isolated from farm soil

The taxonomic position of a novel Gram-negative strain, designated Sy1(T), isolated from a farm-soil sample obtained from Jiangsu Province, PR China, was characterized by using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 degrees C and at pH 6.0-8.0. Based on 16S rRNA gene sequence analysis, strain Sy1(T) is a member of the genus Sphingobacterium; Sphingobacterium multivorum JCM 21156(T) was the nearest relative (98.5% sequence similarity). The predominant fatty acids of strain Sy1T were isoC15:0 (32.90/o), C16:0 (10.9%) and summed feature 3 (iso-C-15:0 2-OH and/or C-16:1 omega 7c; 24.1%). The DNA G + C content was 38.5 mol%. The low level of DNA-DNA relatedness (2.2 %) to S. multivorum JCM 21156 T in combination with differential morphological and biochemical properties demonstrated that strain SY1(T) (=KCTC 22131(T)= CGMCC 1.6855(T)) should be classified as representing a novel species of the genus Sphingobacterium for which the name Sphingobacterium siyangense sp. nov. is proposed.

Flavobacterium anhuiense sp nov., isolated from field soil

A novel strain, D3(T), isolated from a field-soil sample obtained from Anhui Province, PR China, was characterized taxonomically by using a polyphasic approach. The cells were Gram-negative, yellow-pigmented rods devoid of flagella, but showing gliding motility. The organism was able to grow at 5-37 degrees C and at pH 4.0-10.0. A comparative 16S rRNA gene sequence analysis indicated that strain D3(T) is a member of the genus Flavobacterium, sharing highest sequence similarity with the type strain of Flavobacterium defluvii (96.7 %). The major isoprenoid quinone was MK-6 and the predominant fatty acids were iso-C-15:0, summed feature 3 (C-16:1 omega 7c and/or iso-C-15:0 2-OH) and C-16:0. The DNA G + C content was 31.4 mol%. On the basis of phylogenetic and phenotypic data, strain D3(T) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium anhuiense sp. nov. is proposed. The type strain is D3(T) (=KCTC 22128(T)= CGIVICC 1.6859(T)).

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI1 and BMSI2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 mu M and 0.15 mu M, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. (c) 2007 Elsevier Inc. All rights reserved.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR). (C) 2002 Elsevier Science Inc. All rights reserved.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

Characterization of cDNA encoding immunoglobulin light chain of the Mandarin fish (Siniperca chuatsi)

Immunoglobulin light chain cDNA sequences of a perciform fish, the mandarin fish Siniperca chuatsi were amplified from head kidney mRNA by reverse transcription (RT)-PCR and RACE methods using degenerated primer and gene specific ones. In cDNA sequences of the VL region, nucleotide exchanges were present mainly within CDRs, although a lesser degree of variability was also found in FRs. Moreover, the length of CDRI and CDR3 in the mandarin fish is shorter than in most other fish species. In the middle of S. chuatsi CL region, a microsatellite sequence (AGC)(6-8) was found, which is also present in another perciform species, the spotted wolffish (Anarhichas minor). The comparison of amino acid sequence of the mandarin fish CL domain with those of other vertebrates showed the highest degree of similarity of 94.5% to the spotted wolffish, while the similarity with rainbow trout (Oncorhynchus mykiss) Ig L1 (62.7%) and channel catfish (Ictalurus punctatus) Ig LG (55.9%) isotypes is also higher. However, there is only 50% identity in the VL regions between the mandarin fish and the wolffish. The sequence similarity of the mandarin fish CL domain with those of higher vertebrate did not readily allow it to be classified as kappa or lambda isotype. The phylogenetic analyses also demonstrated that the CL genes of the mandarin fish and most other teleost fish cluster as a separate branch out of the mammal kappa and lambda branches. (C) 2003 Elsevier B.V. All rights reserved.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

Wangia profunda gen. nov., sp nov., a novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa Trough deep-sea sediment

An orange-pigmented, Gram-negative, nonmotile, strictly aerobic and oxidase- and catalase-positive bacterium (SM-A87(T)) was isolated from the deep-sea sediment of the southern Okinawa Trough area. The main fatty acids were i15 : 0, i17 : 0 3OH, i15 : 1 G, i17 : 1 omega 9c, 15 : 0, i15 : 0 3OH and summed feature 3 (comprising i-15 : 0 2OH and/or 16 : 1 omega 7c). MK-6 was the predominant respiratory quinone. DNA G+C content was 35.8 mol%. Flexirubin-type pigments were absent. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain SM-A87(T) formed a distinct lineage within the family Flavobacteriaceae, with < 93% sequence similarity to the nearest strain of genus Salegentibacter. Moreover, strain SM-A87(T) could be distinguished from the nearest phylogenetic neighbors by a number of chemotaxonomic and phenotypic properties. On the basis of polyphasic analyses, it is proposed that strain SM-A87(T) be classified in a novel genus and a new species in the family Flavobacteriaceae, designated Wangia profunda gen. nov., sp. nov. The type strain is SM-A87(T) (CCTCC AB 206139(T)=DSM 18752).

Self-similarity limits of genomic signatures

It is shown that metric representation of DNA sequences is one-to-one. By using the metric representation method, suppression of nucleotide strings in the DNA sequences is determined. For a DNA sequence, an optimal string length to display genomic signature in chaos game representation is obtained by eliminating effects of the finite sequence. The optimal string length is further shown as a self-similarity limit in computing information dimension. By using the method, self-similarity limits of bacteria complete genomic signatures are further determined.

NK-lysin of channel catfish: Gene triplication, sequence variation, and expression analysis

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In addition to the previously known four classes of antimicrobial peptides, a fifth class of antimicrobial peptides has been recently identified to include NK-lysins that have a globular three-dimensional structure and are larger with 74-78 amino acid residues. NK-lysin has been shown to harbor antimicrobial activities against a wide spectrum of microorganisms including bacteria, fungi, protozoa, and parasites. To date, NK-lysin genes have been reported from only a limited number of organisms. We previously identified a NK-lysin cDNA in channel catfish. Here we report the identification of two noveltypes of NK-lysin transcripts in channel catfish. Altogether, three distinct NK-lysin transcripts exist in channel catfish. In this work, their encoding genes were identified, sequenced, and characterized. We provide strong evidence that the catfish NK-lysin gene is tripled in the same genomic neighborhood. All three catfish NK-lysin genes are present in the same genomic region and are tightly linked on the same chromosome, as the same BAC clones harbor all three copies of the NK-lysin genes. All three NK-lysin genes are expressed, but exhibit distinct expression profiles in various tissues. In spite of the existence of a single copy of NK-lysin gene in the human genome, and only a single hit from the pufferfish,genome, there are two tripled clusters of NK-lysin genes on chromosome 17 of zebrafish in addition to one more copy on its chromosome 5. The similarity in the genomic arrangement of the tripled NK-lysin genes in channel catfish and zebrafish suggest similar evolution of NK-lysin genes. (c) 2005 Elsevier Ltd. All rights reserved.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.