The limited sequencing approach was used to study the molecular epidemiology of 24 Malaysian dengue 2 viruses which were isolated between 1968 and 1993. The sequences of a 240-nucleotide-long region across the envelope/non-structural 1 protein (E/NS1) gene junction of the isolates were determined and analysed. Alignment and comparison of the nucleotide and deduced amino acid sequences of the isolates revealed that nucleotide changes occurred mostly at the third position of a particular codon and were of the transition (AG, CU) type. Five nucleotide changes resulted in amino acid substitutions. Pairwise comparisons of the nucleotide sequences gave divergence values ranging from 0 to 9.2%. At the amino acid level, the divergence ranged between 0 and 3.8%. Based on the 6% divergence as the cut-off point for genotypic classification, the isolates were grouped into two genotypes, I and II. Comparison of the nucleotide sequences of the Malaysian dengue isolates with those of the dengue viruses of other regions of the world revealed that members of genotypes I and II were closely related to viruses from the Indian Ocean and Western Pacific regions, respectively.
Di-nucleotide microsatellites were isolated from a genomic library of a tropical tree species, Dryobalanops lanceolata, in Sarawak, for the purpose of using them as hypervariable genetic markers to study the pollen-mediated gene flow. Among 1600 recombinant clones, in total 20 clones gave positive signals when hybridized with oligonucleotides with the three different repeat motifs, GT, CA and CT. Estimations of abundance of (GT)n/(CA)n and (GA)n/(CT)n dinucleotide repeats in D. lanceolata genome revealed to be one in every 84 kb and 80 kb, respectively. Among six sequenced microsatellite loci, one was selected to synthesize PCR primers to amplify the microsatellite. PCR product size of the locus was variable among different individuals, which is attributed to the different number of di-nucleotide repeats. The same microsatellite genotype was detected in the trunk and canopy of a single large tree, indicating the utility of trunk tissue as the source of DNA for the population genetic study of tropical tree species, the canopy of which is usually difficult to approach.
Swamp and river buffalo mitochondrial DNA (mtDNA) was sequenced for 303 bp of the cytochrome b gene for 54 animals from 14 populations, and for 158 bp of the D-loop region for 80 animals from 11 populations. Only one cytochrome b haplotype was found in river buffalo. Of the four haplotypes identified in swamp buffalo, one found in all populations is apparently ancestral both to the other swamp haplotypes and to the river haplotype. The phylogenetic relationships among the 33 D-loop haplotypes, with a cluster of 11 found in swamp buffalo only, also support the evolution of domesticated swamp and river buffalo from an ancestral swamp-like animal, most likely represented today by the wild Asian buffalo (Bubalus arnee). The time of divergence of the swamp and river types, estimated from the D-loop data, is 28,000 to 87,000 years ago. We hypothesise that the species originated in mainland south-east Asia, and that it spread north to China and west to the Indian subcontinent, where the rive type evolved and was domesticated. Following domestication in China, the domesticated swamp buffalo spread through two separate routes, through Taiwan and the Philippines to the eastern islands of Borneo and Sulawesi, and south through mainland south-east Asia and then to the western islands of Indonesia.
The xenobiotic metabolizing enzymes N-acetyltransferases (NATs) are important for the biotransformation and/or bioactivation of drugs and carcinogens. NATs are coded for in humans by two distinct genes, designated NAT1 and NAT2. NAT1, which was originally thought to be monomorphic, was recently reported to exhibit variation in human populations. Recent studies suggested that a genetic polymorphism of NAT1 may be associated with colorectal cancer risk. The distributions of NAT1 allele and genotype frequencies in unrelated individuals among Indian (n = 140), Malay (n = 122) and Chinese (n = 181) populations in Singapore were characterized by polymerase chain reaction-restriction fragment length polymorphism and allele-specific-polymerase chain reaction. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Indians were 0.3, 0.51, 0.17 and 0.02, respectively. The corresponding NAT1 allelic frequencies in Malays were 0.29, 0.30, 0.39 and 0.02, respectively, and were similar to those in Chinese in the region. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Chinese were 0.33, 0.35, 0.30 and 0.02, respectively. These findings are of importance for the determination of cancer risk in these populations. In addition, nucleotide changes at positions 350-351 (GG to CC) and 497-499 (GGG to CCC) of the NAT1 gene were not found in the alleles of the populations studied.
The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.
A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.
The beta-thalassaemia mutations in 20 Malaysian children with beta-thalassaemia major were characterised by using a multi-modal approach, consisting of a slot-blot hybridisation with selected allele-specific oligonucleotides (ASO), followed by reverse dot-blot assay (RDB), amplification refractory mutation system (ARMS) and genomic sequencing. This strategy yielded a 94.4% mutation detection rate. The 6 most common mutations were codons 41/42 (-TTCT), IVS II nt 654(C --> T), IVS I nt 5(G --> C), IVS I nt 1(G -->T), codon 35 (-C) and codon 19 (A --> G), which accounted for 83.3% of all mutations detected. A strategy of initial screening with the above 6 selected ASOs for slot-blot hybridisation followed by RDB assay for the less common Asian mutations would give a mutation identification of 91.7%. Another feasible approach would be to analyse alleles from a particular racial group, by a judicious selection of 4 ASOs common to that particular subpopulation and then supplement this with RDB assay. This could yield a 100% coverage for the Chinese subpopulation in Malaysia. With these strategies, a practical approach has been identified to overcome the pitfalls posed by the molecular heterogeneity of beta-thalassaemia to enable prenatal diagnosis and carrier screening to be carried out. Regional collaborative studies are to be encouraged as an indispensable tool in providing better health care services to our patients.
HIV spread in South and South-East Asia is most alarming, and genetic variability of HIV-1 is an important consideration in vaccine development. In this study, we examined the third variable (V3) region of env gene of HIV-1 variants prevalent in Thailand, Malaysia, India, and the Philippines. By phylogenetic tree analyses, an HIV-1 variant from an injecting drug user (IDU) in Thailand belonged to subtype B, and HIV-1 variants from 2 IDUs in Malaysia were classified into 2 subtypes, B and E. One HIV-1 variant from a male homosexual in the Philippines belonged to subtype B. Out of 8 HIV-1 variants from sexually transmitted disease patients in India, 7 belonged to subtype C, and one to subtype A. Although the total number of individuals examined in this study was limited, 4 HIV-1 subtypes were found in South and South-East Asia and large international movements of HIV-1-infected individuals in this region could induce global dissemination of these HIV-1 variants.
The L-amino acid oxidase of Malayan pit viper (Calloselasma rhodostoma) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 132,000 as determined by Sephadex G-200 gel filtration chromatography and 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a glycoprotein, has an isoelectric point of 4.4, and contains 2 mol of flavin mononucleotide per mole of enzyme. The N-terminal amino acid sequence of the enzyme was A-D-D-R-N-P-L-A-E-E-F-Q-E-N-N-Y-E-E-F-L. Kinetic studies suggest the presence of a alkyl side-chain binding site in the enzyme and that the binding site comprises at least four hydrophobic subsites. The characteristics of the binding site differ slightly from those of cobra venom L-amino acid oxidases.
Nucleotide sequences were obtained for the second internal transcribed spacer of the ribosomal gene repeat and for part of the mitochondrial-cytochrome c oxidase subunit I gene from geographical isolates of Paragonimus westermani from Japan, China, Korea, Taiwan, the Philippines, peninsular Malaysia and Thailand. Sequences were obtained from several other species of Paragonimus for comparative purposes. Two groups were recognized within P. westermani: an NE group (China, Japan, Korea, Taiwan) which was relatively uniform and included both diploid and triploid forms, and a southern group (Malaysia, Thailand, Philippines), members of which were genetically distant from one another. According to both ITS2 and COI data, genetic distances among P. westermani isolates equalled or exceeded those between some distinct species of Paragonimus. The ITS2 sequences were conserved relative to COI sequences. Substitutions among the latter may be approaching saturation within the genus Paragonimus.
Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.
Members of the Semai group of Orang Asli ('aborigines') in peninsular Malaysia were examined for apolipoprotein E (apo E) variants in relation to plasma total cholesterol (TC), high density lipoprotein cholesterol, low density lipoprotein cholesterol (LDLC), triglycerides (TG), apolipoprotein AI and apolipoprotein B (apo B). The e2 and e4 alleles were found to be higher than in most other groups as reported. The sample as a whole was normotriglyceridaemic (mean plasma TG, 1.5 mmol/l) and very markedly hypocholesterolaemic (mean plasma TC 1.7 mmol/l). The distribution of apo E variants was not related to any of the plasma lipids or apolipoprotein fractions using results from all subjects, but if a distinctly hypertriglyceridaemic sub-section was omitted (TG > 1.7 mmol/l) then apo E variants were determinants of plasma TC, LDLC, and apo B concentrations, the lower values of these being associated with the 2-2 and 2-3 genotypes, and the higher with 3-4, and 4-4.
Rice tungro spherical virus (RTSV) has an RNA genome of more than 12 kb with various features which classify it as a plant picornavirus. The capsid comprises three coat protein (CP) species, CP1, CP2 and CP3, with predicted molecular masses of 22.5, 22.0 and 33 kDa, respectively, which are cleaved from a polyprotein. In order to obtain information on the properties of these proteins, each was expressed in E. coli, purified as a fusion to the maltose-binding protein and used for raising a polyclonal antiserum. CP1, CP2 and CP3 with the expected molecular masses were detected specifically in virus preparations. CP3 is probably the major antigenic determinant on the surface of RTSV particles, as was shown by ELISA, Western blotting and immunogold electron microscopy using antisera obtained against whole virus particles and to each CP separately. In some cases, especially in crude extracts, CP3 antiserum detected several other proteins (40-42 kDa), which could be products of CP3 post-translational modification. No serological differences were detected between the three CPs from isolates from the Philippines, Thailand, Malaysia and India. The CP3-related 40-42 kDa proteins of the Indian RTSV isolate have a slightly higher electrophoretic mobility (42-44 kDa) and a different response to cellulolytic enzyme preparations, which allows them to be differentiated from south-east Asian isolates.
A novel derivative of sucrose, beta-(3,6-di-O-feruloyl)-fructofuranosyl-alpha-(2,3,4,6-tetra-O-ac etyl)- glucopyranoside, was isolated from the wood of Bhesa paniculata. Its structure was determined by a combination of 2D 1H-1H and 1H-13C correlation NMR spectroscopy. The known compounds, glycerol 1-9',12'-octadecadienoate, beta-sitosterol, (+/-)-pinoresinol, methyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxybenzoic acid, anofinic acid and 2-(1'-methylethenyl)-benzofuran-5-carboxylic acid were also isolated.
1. Bradykinin and related kinins may act on four types of receptors designated as B1, B2, B3 and B4. It seems that the B2 receptors are most commonly found in various vascular and non-vascular smooth muscles, whereas B1 receptors are formed in vitro during trauma, and injury, and are found in bone tissues. 2. These BK receptors are involved in the regulations of various physiological and pathological processes. 3. The mode of kinin actions are based upon the interactions between the kinin and their specific receptors, which can lead to activation of several second-messenger systems. 4. Recently, numerous BK receptors antagonists have been synthesized with prime aim to treat diseases caused by excessive kinin production. 5. These diseases are RA, inflammatory diseases of the bowel, asthma, rhinitis and sore throat, allergic reactions, pain, inflammatory skin disorders, endotoxin and anaphylactic shock and coronary heart diseases. 6. On the other hand, BK receptor antagonists could be contraindicated in hypertension, since these drugs may antagonize the antihypertensive therapy and/or may trigger the hypertensive crisis. 7. It is worth suggesting that the BK receptor agonists might be useful antihypertensive drugs.
The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.
We investigate the evolution of host association in a cryptic complex of mutualistic Crematogaster (Decacrema) ants that inhabits and defends Macaranga trees in Southeast Asia. Previous phylogenetic studies based on limited samplings of Decacrema present conflicting reconstructions of the evolutionary history of the association, inferring both cospeciation and the predominance of host shifts. We use cytochrome oxidase I (COI) to reconstruct phylogenetic relationships in a comprehensive sampling of the Decacrema inhabitants of Macaranga. Using a published Macaranga phylogeny, we test whether the ants and plants have cospeciated. The COI phylogeny reveals 10 well-supported lineages and an absence of cospeciation. Host shifts, however, have been constrained by stem traits that are themselves correlated with Macaranga phylogeny. Earlier lineages of Decacrema exclusively inhabit waxy stems, a basal state in the Pachystemon clade within Macaranga, whereas younger species of Pachystemon, characterized by nonwaxy stems, are inhabited only by younger lineages of Decacrema. Despite the absence of cospeciation, the correlated succession of stem texture in both phylogenies suggests that Decacrema and Pachystemon have diversified in association, or codiversified. Subsequent to the colonization of the Pachystemon clade, Decacrema expanded onto a second clade within Macaranga, inducing the development of myrmecophytism in the Pruinosae group. Confinement to the aseasonal wet climate zone of western Malesia suggests myrmecophytic Macaranga are no older than the wet forest community in Southeast Asia, estimated to be about 20 million years old (early Miocene). Our calculation of COI divergence rates from several published arthropod studies that relied on tenable calibrations indicates a generally conserved rate of approximately 1.5% per million years. Applying this rate to a rate-smoothed Bayesian chronogram of the ants, the Decacrema from Macaranga are inferred to be at least 12 million years old (mid-Miocene). However, using the extremes of rate variation in COI produces an age as recent as 6 million years. Our inferred timeline based on 1.5% per million years concurs with independent biogeographical events in the region reconstructed from palynological data, thus suggesting that the evolutionary histories of Decacrema and their Pachystemon hosts have been contemporaneous since the mid-Miocene. The evolution of myrmecophytism enabled Macaranga to radiate into enemy-free space, while the ants' diversification has been shaped by stem traits, host specialization, and geographic factors. We discuss the possibility that the ancient and exclusive association between Decacrema and Macaranga was facilitated by an impoverished diversity of myrmecophytes and phytoecious (obligately plant inhabiting) ants in the region.
Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.
Alcanivorax hongdengensis A-11-3 is a newly identified type strain isolated from the surface water of the Malacca and Singapore Straits that can degrade a wide range of alkanes. To understand the degradation mechanism of this strain, the genes encoding alkane hydroxylases were obtained by PCR screening and shotgun sequencing of a genomic fosmid library. Six genes involved in alkane degradation were found, including alkB1, alkB2, p450-1, p450-2, p450-3 and almA. Heterogeneous expression analysis confirmed their functions as alkane oxidases in Pseudomonas putida GPo12 (pGEc47ΔB) or Pseudomonas fluorescens KOB2Δ1. Q-PCR revealed that the transcription of alkB1 and alkB2 was enhanced in the presence of n-alkanes C(12) to C(24); three p450 genes were up-regulated by C(8)-C(16) n-alkanes at different levels, whereas enhanced expression of almA was observed when strain A-11-3 grew with long-chain alkanes (C(24) to C(36)). In the case of branched alkanes, pristane significantly enhanced the expression of alkB1, p450-3 and almA. The six genes enable strain A-11-3 to degrade short (C(8)) to long (C(36)) alkanes that are straight or branched. The ability of A. hongdengensis A-11-3 to thrive in oil-polluted marine environments may be due to this strain's multiple systems for alkane degradation and its range of substrates.