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).
During a study on the quality of the indoor environment, Acanthamoeba spp. were detected in 20 out of 87 dust samples collected from air-conditioners installed in a four-story campus building located in Kuala Lumpur, Malaysia. Twenty-one cloned Acanthamoeba isolates designated as IMU1 to IMU21 were established from the positive primary cultures. Five species were identified from the 16 isolates according to the morphological criteria of Pussard and Pons; i.e. A. castellanii, A. culbertsoni, A. griffini, A. hatchetti and A. polyphaga. Species identities for the remaining five isolates (IMU4, IMU5, IMU15, IMU20 and IMU21), however, could not be determined morphologically. At genotypic characterization, these isolates were placed into T3 (IMU14); T5 (IMU16 and IMU17) and T4 (all the remaining isolates). To predict the potential pathogenicity of these Acanthamoeba isolates, thermo- and osmotolerance tests were employed; many isolates were predicted as potential human pathogens based on the outcome of these tests. This is the first time potentially pathogenic Acanthamoeba have been isolated from air-conditioners in Malaysia.
A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg(-1) which corresponds to 2927.15 Ug(-1) of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65 degrees C and pH 9, respectively. It was stable up to 65 degrees C at pH 7 and active over a wide pH range (pH 6-11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
The FOXE1 gene was screened for mutations in a cohort of 34 unrelated patients with congenital hypothyroidism, 14 of whom had thyroid dysgenesis and 18 were normal (the thyroid status for 2 patients was unknown). The entire coding region of the FOXE1 gene was PCR-amplified, then analyzed using single-stranded conformational polymorphism, followed by confirmation by direct DNA sequencing. DNA sequencing analysis revealed a heterozygous A>G transition at nucleotide position 394 in one of the patients. The nucleotide transition changed asparagine to aspartate at codon 132 in the highly conserved region of the forkhead DNA binding domain of the FOXE1 gene. This mutation was not detected in a total of 104 normal healthy individuals screened. The binding ability of the mutant FOXE1 protein to the human thyroperoxidase (TPO) promoter was slightly reduced compared with the wild-type FOXE1. The mutation also caused a 5% loss of TPO transcriptional activity.
The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape.
The relationship between a putative metallothionein gene (MT) and exposure to cadmium (Cd) in blood cockles (Anadara granosa) is reported. In a 96-h dose-response experiment, mortality of cockles was found to proportionately increase in the range of 0.2-5.0 mg/l Cd with a calculated LC(50) of 2.94 mg/l. Exposure to 0.25 mg/l Cd for 16 days caused significant increases (P<0.05) in Cd concentrations in whole tissues, gills and hepatopancreas, and the accumulation of Cd in these tissues increased with the duration of exposure. Two cDNA libraries constructed using the hepatopancreas from control and Cd-treated cockles gave titres of 5.62 x 10(5) and 1.94 x 10(5) pfu/microg vector, respectively. A putative MT gene, AnaMT, of 510 nucleotides in length, was isolated from the treated cDNA library using a heterologous probe MT20 from the blue mussel, Mytilus edulis. Northern analyses using AnaMT as a probe indicated low expression of the MT mRNA in control animals. In cockles treated with 0.25 mg/l Cd for 4 days, MT mRNA level increased to approximately 168%, but declined to 108% at day 8. After 12 and 16 days of Cd treatment, expression of the MT gene was 138% and 187%, respectively, compared to the controls. These observations suggest that induction of the MT gene by a sublethal dose of Cd is rapid, occurring within 4 days of treatment.
Zebrafish possess two isoforms of vertebrate ancient long (VAL)-opsin, val-opsinA (valopa) and val-opsinB (valopb), which probably mediate non-visual responses to light. To understand the diurnal and light-sensitive regulation of the valop genes in different cell groups, the current study used real-time quantitative PCR to examine the diurnal changes of valopa and b mRNA levels in different brain areas of adult male zebrafish. Furthermore, effects of the extended exposure to light or dark condition, luminous levels and the treatment with a melatonin receptor agonist or antagonist on valop transcription were examined. In the thalamus, valop mRNA levels showed significant diurnal changes; valopa peaked in the evening, while valopb peaked in the morning. The diurnal change of valopa mRNA levels occurred independent of light conditions, whereas that of valopb mRNA levels were regulated by light. A melatonin receptor agonist or antagonist did not affect the changes of valop mRNA levels. In contrast, the midbrain and hindbrain showed arrhythmic valop mRNA levels under light and dark cycles. The differential diurnal regulation of the valopa and b genes in the thalamus and the arrhythmic expression in the midbrain and hindbrain suggest involvement of deep brain VAL-opsin in time- and light-dependent physiology. We show diurnal expression changes of vertebrate ancient long (VAL) opsin genes (valopa and valopb), depending on brain area, time of day and light condition, in the adult male zebrafish. Differential regulation of the valop genes in the thalamus and arrhythmic expression in the midbrain and hindbrain suggest their involvement in time- and light-dependent physiology to adjust to environmental changes.
The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA--random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.
Lactic acid bacteria (LAB) can be isolated from traditional milk products. LAB that secrete substances that inhibit pathogenic bacteria and are resistant to acid, bile, and pepsin but not vancomycin may have potential in food applications.
Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.
Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14 415 bp), S. japonicum (Anhui strain, China; 14 085 bp) and S. mekongi (Khong Island, Laos; 14 072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 aa), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79% for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species.
Random amplification of polymorphic DNA (RAPD) was used to analyse genomic DNA from virgin females and males of Brugia malayi, with a view to identifying sex-specific differences predicted by an XX/XY system of chromosomal sex determination. A product of 2338 bp, amplified with the arbitrary primer 5' GTTGCGATCC 3', was obtained exclusively from males. Primers based on the sequence of this product amplified a DNA fragment of the expected size from each of two independent isolates of B. malayi (from Malaysia and Indonesia) by PCR. No reaction product was obtained from the closely related species Brugia pahangi. In a genetic cross between B. malayi males and B. pahangi females, F1 hybrid microfilariae were PCR-positive, indicating that the locus is paternally-inherited. Southern blotting demonstrated that the target sequence resides in the high molecular weight fraction of genomic DNA, confirming that it is of chromosomal, rather than mitochondrial, origin. Sequencing of the locus revealed significant similarity with members of a family of reverse transcriptase-like genes in Caenorhabditis elegans. In-frame stops indicate that the gene is non-functional, but multiple bands of hybridisation in Southern blots suggest that the RT sequence may be the relic of a transposable element. Multiple repeats of the dinucleotide AT occurred in another region of the sequence. These varied in number between the two isolates of B. malayi in the manner of a microsatellite, surprisingly the first to be described from the B. malayi genome. Because of its association with the Y chromosome, we have given the locus the acronym TOY (Tag On Y). Identification of this chromosome-specific marker confirms the XX/XY heterogametic karyotype in B. malayi and opens the way to elucidation of the role of Y in sex determination.
Coptotermes curvignathus Holmgren is capable of feeding on living trees. This ability is attributed to their effective digestive system that is furnished by the termite's own cellulolytic enzymes and cooperative enzymes produced by their gut microbes. In this study, the identity of an array of diverse microbes residing in the gut of C. curvignathus was revealed by sequencing the near-full-length 16S rRNA genes. A total of 154 bacterial phylotypes were found. The Bacteroidetes was the most abundant phylum and accounted for about 65% of the gut microbial profile. This is followed by Firmicutes, Actinobacteria, Spirochetes, Proteobacteria, TM7, Deferribacteres, Planctomycetes, Verrucomicrobia, and Termite Group 1. Based on the phylogenetic study, this symbiosis can be a result of long coevolution of gut enterotypes with the phylogenic distribution, strong selection pressure in the gut, and other speculative pressures that determine bacterial biome to follow. The phylogenetic distribution of cloned rRNA genes in the bacterial domain that was considerably different from other termite reflects the strong selection pressures in the gut where a proportional composition of gut microbiome of C. curvignathus has established. The selection pressures could be linked to the unique diet preference of C. curvignathus that profoundly feeds on living trees. The delicate gut microbiome composition may provide available nutrients to the host as well as potential protection against opportunistic pathogen.