A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of >or=87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I-IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China.
The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.
Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.
Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 A. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 A away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.
Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.
The coding region of guinea pig peroxisome proliferator activated receptor gamma1 (gpPPARgamma1) cDNA was successfully cloned from adipose tissue by reverse transcription polymerase chain reaction (RT-PCR) using the designated primers based on the conserved regions of the other mammalian PPARgamma1 sequence. From RT-PCR, a combination of three cDNA fragments that comprised of the full length coding region PPARgamma1 cDNA gene were amplified, with the size of 498, 550 and 557 bp, respectively. All three fragments were then successfully assembled by utilising the internal restriction sites present at the overlapping regions to give rise to the full-length coding region of gpPPARgamma1 with the size of 1428 bp and consisting of 475 amino acids. Guinea pig PPARgamma1 is highly conserved with those of other species at protein and nucleotide levels. Gene expression studies showed that gpPPARgamma mRNA was predominantly expressed in adipose tissue followed by lung and spleen. However, at the protein level, PPARgamma was also found to be expressed in skeletal muscle.
A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.
The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.
Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.
The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.
Bungarus fasciatus is one of three species of krait found in Malaysia. Envenoming by B. fasciatus results in neurotoxicity due to the presence of presynaptic and postsynaptic neurotoxins. Antivenom, either monovalent or polyvalent, is the treatment of choice in systemically envenomed patients. In this study, we have isolated a postsynaptic neurotoxin which we named α-elapitoxin-Bf1b. This toxin has an approximate molecular weight of 6.9 kDa, with LCMS/MS data showing that it is highly homologous with Neurotoxin 3FTx-RI, a toxin identified in the Bungarus fasciatus venom gland transcriptome. α-Elapitoxin-Bf1b also shared similarity with short-chain neurotoxins from Laticauda colubrina and Pseudechis australis. α-Elapitoxin-Bf1b produced concentration- and time-dependent neurotoxicity in the indirectly-stimulated chick biventer cervicis muscle preparation, an effect partially reversible by repetitive washing of the preparation. The pA2 value for α-elapitoxin-Bf1b of 9.17 ± 0.64, determined by examining the effects of the toxin on cumulative carbacol concentration-response curves, indicated that the toxin is more potent than tubocurarine and α-bungarotoxin. Pre-incubation of Bungarus fasciatus monovalent and neuro polyvalent antivenom failed to prevent the neurotoxic effects of α-elapitoxin-Bf1b in the chick biventer cervicis muscle preparation. In conclusion, the isolation of a postsynaptic neurotoxin that cannot be neutralized by either monovalent and polyvalent antivenoms may indicate the presence of isoforms of postsynaptic neurotoxins in Malaysian B. fasciatus venom.
The dominant factors controlling soil bacterial community variation within the tropics are poorly known. We sampled soils across a range of land use types--primary (unlogged) and logged forests and crop and pasture lands in Malaysia. PCR-amplified soil DNA for the bacterial 16S rRNA gene targeting the V1-V3 region was pyrosequenced using the 454 Roche machine. We found that land use in itself has a weak but significant effect on the bacterial community composition. However, bacterial community composition and diversity was strongly correlated with soil properties, especially soil pH, total carbon, and C/N ratio. Soil pH was the best predictor of bacterial community composition and diversity across the various land use types, with the highest diversity close to neutral pH values. In addition, variation in phylogenetic structure of dominant lineages (Alphaproteobacteria, Beta/Gammaproteobacteria, Acidobacteria, and Actinobacteria) is also significantly correlated with soil pH. Together, these results confirm the importance of soil pH in structuring soil bacterial communities in Southeast Asia. Our results also suggest that unlike the general diversity pattern found for larger organisms, primary tropical forest is no richer in operational taxonomic units of soil bacteria than logged forest, and agricultural land (crop and pasture) is actually richer than primary forest, partly due to selection of more fertile soils that have higher pH for agriculture and the effects of soil liming raising pH.
It is known that the microbial community of the rhizosphere is not only influenced by factors such as root exudates, phenology, and nutrient uptake but also by the plant species. However, studies of bacterial communities associated with tropical rainforest tree root surfaces, or rhizoplane, are lacking. Here, we analyzed the bacterial community of root surfaces of four species of native trees, Agathis borneensis, Dipterocarpus kerrii, Dyera costulata, and Gnetum gnemon, and nearby bulk soils, in a rainforest arboretum in Malaysia, using 454 pyrosequencing of the 16S rRNA gene. The rhizoplane bacterial communities for each of the four tree species sampled clustered separately from one another on an ordination, suggesting that these assemblages are linked to chemical and biological characteristics of the host or possibly to the mycorrhizal fungi present. Bacterial communities of the rhizoplane had various similarities to surrounding bulk soils. Acidobacteria, Alphaproteobacteria, and Betaproteobacteria were dominant in rhizoplane communities and in bulk soils from the same depth (0-10 cm). In contrast, the relative abundance of certain bacterial lineages on the rhizoplane was different from that in bulk soils: Bacteroidetes and Betaproteobacteria, which are known as copiotrophs, were much more abundant in the rhizoplane in comparison to bulk soil. At the genus level, Burkholderia, Acidobacterium, Dyella, and Edaphobacter were more abundant in the rhizoplane. Burkholderia, which are known as both pathogens and mutualists of plants, were especially abundant on the rhizoplane of all tree species sampled. The Burkholderia species present included known mutualists of tropical crops and also known N fixers. The host-specific character of tropical tree rhizoplane bacterial communities may have implications for understanding nutrient cycling, recruitment, and structuring of tree species diversity in tropical forests. Such understanding may prove to be useful in both tropical forestry and conservation.
Previously we have shown that avian leukosis virus subgroup J (ALV-J) might be present in chicken flocks from Malaysia based on serological study and also on detection of tissue samples with myelocytic infiltration. In this study, the polymerase chain reaction was used to detect ALV-J sequences from archived frozen samples. Out of 21 tissue samples examined, 16 samples were positive for proviral DNA and four samples for ALV-J RNA. However, only nine samples were found positive for myelocytic infiltration. A total of 465 base pairs equivalent to positions 5305 to 5769 of HPRS-103 from each of the viral RNA positive samples were characterized. Sequence analysis indicated that the samples showed high identity (95.9 to 98.2%) and were close to HPRS-103 with identities between 97.4 and 99.3%. This study indicates that ALV-J-specific sequences can be detected by polymerase chain reaction from frozen tissue samples with and without myelocytic infiltration.
To assess the prevalence of major drug resistance mutations in antiretroviral (ARV)-treated patients with detectable viral load (VL) in Kuala Lumpur, Malaysia, genotypic resistance testing was performed among treated human immunodeficiency virus type 1 (HIV-1) patients attending the University Malaya Medical Center between July 2003 and November 2004. The reverse transcriptase (RT) and protease genes from 36 plasma samples with detectable VL were examined for major mutations associated with ARV resistance as reported by the International AIDS Society-USA Drug Resistance Mutations Group. The prevalence of patients with at least one major mutation conferring drug resistance to nucleoside RT inhibitors (NRTIs), non-NRTIs (NNRTIs) or protease inhibitors (PIs) was 77.8%. In the RT gene, the frequency of mutations associated with NRTIs and NNRTIs resistance was 52.8 and 63.9%, respectively, with M184V and K103N mutations being selected most frequently by these drugs. A patient with Q151M mutation complex was also detected. Twenty-two percent of the patients had mutations associated with PIs. The following pattern of prevalence of ARV-resistant HIV-1 variants was observed: NNRTI-resistant > NRTI-resistant > PI-resistant. The prevalence of major drug resistance mutations among ARV-treated patients with detectable VL is high in Kuala Lumpur. Genotypic drug resistance testing is therefore important for monitoring patients experiencing ARV regimen failure.
Recent developments in the target based cancer therapies have identified HSF1 as a novel non oncogenic drug target. The present study delineates the design and molecular docking evaluation of Rohinitib (RHT) - Cantharidin (CLA) based novel HSF1 inhibitors for target-based cancer therapy. Here, we exploited the pharmacophoric features of both the parent ligands for the design of novel hybrid HSF1 inhibitors. The RHT-CLA ligands were designed and characterized for ADME/Tox features, interaction with HSF1 DNA binding domain and their pharmacophoric features essential for interaction. From the results, amino acid residues Ala17, Phe61, His63, Asn65, Ser68, Arg71 and Gln72 were found crucial for HSF1 interaction with the Heat shock elements (HSE). The hybrid ligands had better affinity towards the HSF1 DNA binding domain, in comparison to RHT or CLA and interacted with most of the active site residues. Additionally, the HSF1-ligand complex had a reduced affinity towards HSE in comparison to native HSF1. Based on the results, ligand RC15 and RC17 were non carcinogenic, non mutagenic, completely biodegradable under aerobic conditions, had better affinity for HSF1 (1.132 and 1.129 folds increase respectively) and diminished the interaction of HSF1 with HSE (1.203 and 1.239 folds decrease respectively). The simulation analysis also suggested that the ligands formed a stable complex with HSF1, restraining the movement of active site residues. In conclusion, RHT-CLA hybrid ligands can be used as a potential inhibitor of HSF1 for non-oncogene target based cancer therapy.
The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values.
As well as for chemical and environmental reasons, acrylamide is widely used in many industrial applications. Due to its carcinogenicity and toxicity, its discharge into the environment causes adverse effects on humans and ecology alike. In this study, a novel acrylamide-degrading yeast has been isolated. The isolate was identified as Rhodotorula sp. strain MBH23 using ITS rRNA analysis. The results showed that the best carbon source for growth was glucose at 1.0% (w/v). The optimum acrylamide concentration, being a nitrogen source for cellular growth, was at 500 mg l(-1). The highest tolerable concentration of acrylamide was 1500 mg l(-1) whereas growth was completely inhibited at 2000 mg l(-1). At 500 mg l(-1), the strain MBH completely degraded acrylamide on day 5. Acrylic acid as a metabolite was detected in the media. Strain MBH23 grew well between pH 6.0 and 8.0 and between 27 and 30 °C. Amides such as 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, and propionamide supported growth. Toxic heavy metals such as mercury, chromium, and cadmium inhibited growth on acrylamide.
A gene encoding an organic solvent-stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX-4T-1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 degrees C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli.
Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).