Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.
Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979-2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an 'eastern' (BTV-9, -16 and -1) and a 'western' (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.
The NOD2/CARD15 gene has been identified as an important susceptibility gene for Crohn's disease (CD) but the three common disease predisposing mutations (DPM) found in developed countries have not been identified in Asian populations. The aim of our study was to look for the DPM in our multiracial population and to discover whether there were any differences in the three major ethnic groups; Malay, Chinese and Indian.
Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.
The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.
Two Malaysian infectious bronchitis virus isolates, MH5365/95 and V9/04 were characterized based on sequence and phylogenetic analyses of S1, S2, M, and N genes. Nucleotide sequence alignments revealed many point mutations, short deletions, and insertions in S1 region of both IBV isolates. Phylogenetic analysis of S1 gene and sequences analysis of M gene indicated that MH5365/95 and V9/04 belong to non-Massachusetts strain. However, both isolates share only 77% identity. Analysis based on S1 gene showed that MH5365/95 shared more than 87% identity to several Chinese strains. Meanwhile, V9/04 showed only 67-77% identity to all the previously studied IBV strains included in this study suggesting it is a variant of IBV isolate that is unique to Malaysia. Phylogenetic analysis suggests, although both isolates were isolated 10 years apart from different states in Malaysia, they shared a common origin. Analysis based on S2 and N genes indicated that both strains are highly related to each other, and there are fewer mutations which occurred in the respective genes.
Human adipose-derived stem cells (HADSC) have demonstrated the capacity of differentiating into bone depending on the specific induction stimuli and growth factors. However, investigation on stem cell characteristic after osteogenic differentiation is still lacking. The goal of this study was to investigate the differential expression of sternness and osteogenic genes in non-induced HADSC compared with HADSC after osteogenic induction using quantitative Real Time RT-PCR. Our results showed that OCT-4, REX-1, FZD9, OSC, RUNX, and ALP were up regulated after osteogenic induction. This may indicated that HADSCs after osteogenic induction still possessed some stemness properties.
Obtaining pure population of neural cells from embryonic stem (ES) cells remains a challenge as little is known about the genes that govern embryonic stem cell differentiation. Using mouse ES cells, we aim to uncover the mechanisms that regulate neural differentiation of ES cells by focusing on roles played by Wnt family genes. Combining two techniques, Cre/loxP-based genetic recombination and ligand-dependent activation of Cre, we have generated transgenic ES cell lines that allow for the temporal control of expression and activity of Wnt gene (Wnt1-Ha) and Wnt antagonist (Dkk1). The ability of these cell lines in inducing the expression of transgene in undifferentiated ES cells and, more importantly, in differentiated derivatives of ES cells in vitro is evaluated.
Matched MeSH terms: Intercellular Signaling Peptides and Proteins/genetics; Wnt Proteins/genetics
The p53 gene is a tumour suppressor gene that encodes a 393-amino-acid nuclear DNA-binding phosphoprotein. The significance of p53 detection is that p53 mutation is linked with chemo-resistance and transformation to more aggressive disease in a large number of tumour types and it was confirmed that mutant p53 is involved in neoplastic transformations. In addition, the expression of p53 has been closely correlated with clinicopathological findings. Since breast cancer has been reported as one of the most frequent malignancies in women in Malaysia, the expression of p53 was studied in 382 cases of invasive ductal carcinoma of the breast, obtained from three major hospitals in the North-East States of Malaysia. The study utilized an enzyme immunohistochemistry assay for the detection of p53. It was found that p53 was expressed in 29.6% of all the study cases. Furthermore, its expression was significantly correlated with the age and the clinical grading of the disease. No significant statistical correlations were depicted with lymph node status, tumour size, side of tumour, and expression of estrogen and progesterone receptors. Nevertheless, knowledge of the p53 status may be valuable in making clinical decisions regarding diagnosis, prognosis and therapy.
Matched MeSH terms: Breast Neoplasms/genetics*; Carcinoma, Ductal, Breast/genetics*
A novel human leukocyte antigen-B allele officially named B*3589, was found in an indigenous individual of Jehai ethnicity when sequencing was performed to investigate human genome variation in a research project. B*3589 differs form B*3505 in a point mutation at codon 169 (CGC to TGC) resulting in an amino acid change from Arg to Cys.
Matched MeSH terms: HLA-B Antigens/genetics*; Asian Continental Ancestry Group/genetics
In the present study, samples of pond water, sediment and farmed species were collected at 12 fish and shrimp farms in Malaysia, Thailand and Vietnam to determine the biodiversity and environmental distribution of chloramphenicol-resistant (CmR) mesophilic heterotrophs in Southeast Asian aquaculture sites. Following isolation on Iso-Sensitest agar supplemented with 35mug ml(-1) Cm and dereplication using (GTG)(5)-PCR fingerprinting, 557 genotypically unique CmR strains were subjected to polyphasic identification. The 557 mesophilic heterotrophic CmR isolates represented 18 different genera largely dominated by the genera Escherichia (40.2%), Pseudomonas (11.7%), Acinetobacter (11.1%), Klebsiella (7.5%) and Bacillus (5.9%). A total of 439 CmR isolates were further assigned to 31 described species or species groups, mainly including organisms that have been associated with various human opportunistic infections such as Escherichia coli (n=219), Pseudomonas putida (n=47), Klebsiella pneumoniae (n=38) and Acinetobacter baumannii (n=23). Strains of Escherichia, and most notably, of E. coli, were the only common group of CmR heterotrophs irrespective of country, sample type or farm type. Together with other predominant but less widespread groups such as acinetobacters and pseudomonads, the results of this biodiversity study suggest that E. coli can be regarded as a potential indicator of Cm resistance in Southeast Asian aquaculture environments.
Matched MeSH terms: Bacteria/genetics; Drug Resistance, Microbial/genetics
We examined allozyme variation in two camaenid tree snails, Amphidromus atricallosus and A. inversus, across two principal regions of Thailand and from Singapore, plus for A. inversus, one site in peninsular Malaysia. Using horizontal starch gel electrophoresis, 13 allozyme loci (11 polymorphic) were screened for A. atricallosus and 18 (5 polymorphic) for A. inversus. Heterozygosity was higher in A. atricallosus (Hexp=0.018-0.201, mean=0.085) than in A. inversus (Hexp=0-0.023, mean= 0.002). Genetic heterogeneity among samples was higher in A. inversus (Fst=0.965) than in A. atricallosus (Fst=0.781). Within A. atricallosus, populations were more differentiated in southern Thailand (Fst=0.551) than in eastern Thailand (Fst=0.144). The high Fst and low Hexp in populations of A. inversus suggest that this species is likely to have experienced a series of strong bottlenecks, perhaps occurring chiefly on offshore continental-shelf islands. The low Fst values of A. atricallosus in eastern Thailand suggest frequent gene flows among populations in this region. The southern and eastern samples of A. atricallosus exhibited fixed allele differences at four loci and great genetic distance (Nei's D=0.485-0.946), suggesting that these two samples may actually represent, or else be evolving into, separate species.
Two forms of Staurois that are differentiated by body size occur parapatrically in the Crocker Range, Sabah, Borneo. Analyses of a total of 1,499 bp of the mitochondrial cytochrome b, 12S rRNA, and 16S rRNA genes revealed that the two forms could be completely split genetically. The two forms could be also clearly differentiated morphologically, not only by snout-vent length but also by the relative sizes of snout, eye, and finger disk. Comparisons of the two forms with all known species of the genus revealed the large and small forms to be S. tuberilinguis and S. parvus, respectively. The latter species has long been synonymized with the former, but we here consider them to represent different species.
The molecular basis of variable phenotypes in P-thalassaemia patients with identical genotypes has been associated with co-inheritance of alpha-thalassaemia and persistence of HbF production in adult life. The Xmn I restriction site at -158 position of the Ggamma-gene is associated with increased expression of the Ggamma-globin gene and higher production of HbF This study aims to determine the frequency of the digammaferent genotypes of the Ggamma Xmn I polymorphism in P-thalassaemia patients in two ethnic groups in Malaysia. Molecular characterisation and frequency of the Ggamma Xmn I polymorphism were studied in fifty-eight Chinese and forty-nine beta-thalassaemia Malay patients by Xmn I digestion after DNA amplification of a 650 bp sequence. The in-house developed technique did not require further purification or concentration of amplified DNA before restriction enzyme digestion. The cheaper Seakem LE agarose was used instead of Nusieve agarose and distinct well separated bands were observed. Genotyping showed that the most frequent genotype observed in the Malaysian Chinese was homozygosity for the absence of the Xmn I site (-/-) (89.7%). In the Malays, heterozygosity of the Xmn I site (+/-) was most common (63.3%). Homozygosity for the Xmn I site (+/+) was absent in the Chinese, but was confirmed in 8.2% of the Malays. The ratio of the (+) allele (presence of the Xmn I site) to the (-) allele (absence of the Xmn I site)) was higher in the Malays (0.66) compared to the Chinese (0.05). The (+/-) and (+/+) genotypes are more commonly observed in the Malays than the Chinese in Malaysia.
Matched MeSH terms: Deoxyribonucleases, Type II Site-Specific/genetics*; beta-Thalassemia/genetics*
Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of Toxocara canis, Toxocara cati, Toxocara malaysiensis and Toxascaris leonina, specific forward primers were designed in the ITS-1 or ITS-2 for each of the four ascaridoid species of dogs and cats. These primers were used individually together with a conserved primer in the large subunit of rDNA to amplify partial ITS-1 and/or ITS-2 of rDNA from 107 DNA samples from ascaridoids from dogs and cats in China, Australia, Malaysia, England and the Netherlands. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amounts of DNA detectable using the PCR assays were 0.13-0.54ng. These PCR assays should provide useful tools for the diagnosis and molecular epidemiological investigations of toxocariasis in humans and animals.
OBJECTIVE:
To assess the relationship between the HLA-DRB1 genes with disease severity as assessed by radiological erosions in Malaysian patients with rheumatoid arthritis (RA).
METHODS:
In this cross-sectional study, we studied 61 RA patients who fulfilled the ACR criteria for the diagnosis of RA. HLA-DRB1 genotyping was performed by sequence specific primer (SSP) - PCR. Radiological grading and erosive score of the hands and wrists was calculated according to the Larsen-Dale method. Demographic data and treatment given to the patients were obtained from their case records.
RESULTS:
Fifty-six females and five males were studied from three ethnic groups. In 57 patients with erosions, rheumatoid factor was detected in 80%, HLA-DR4 in 40%, HLA-DRB1*0405 in 24% and shared epitope (SE) in 31%. The median delay in starting DMARDs was 24 months. The presence of rheumatoid factor, HLA-DR4 and HLA-DRB1*0405 were not significantly associated with a worse erosive score. Patients who possessed the SE had a higher erosive scores, compared to those who did not (p = 0.05). Concurrently, a delay in starting DMARD was associated with a high erosive score (p = 0.023, r = 0.348). However, after adjustment for the delay in starting DMARD, SE was no longer significantly associated with the erosive score.
CONCLUSIONS:
In these patients, the delay in starting DMARDs had a greater influence on the erosive score than SE alone. Whilst we cannot discount the contribution of the SE presence, we would advocate early usage of DMARDs in every RA patient to reduce joint erosions and future disability.
The mitochondrial DNA control region of the sun bear (Helarctos malayanus) was sequenced using 21 DNA samples collected from confiscated sun bears to identify conservation units, such as evolutionarily significant units and management units, in Sarawak, Borneo Island. A total of 10 haplotypes were observed, indicating the presence of at least two lineages in the sun bear population in Sarawak. Presumably, these two lineages could represent evolutionarily significant units. However, the geographical distributions of the two lineages remained unknown due to the lack of information regarding the exact capture locations of the confiscated sun bears. It is essential to elucidate the geographical distributions of these lineages in order to create a proper conservation plan for the sun bears in Sarawak. Therefore, further studies examining the haplotype distributions using DNA samples from known localities are essential.
A Malaysian family with congenital insensitivity to pain with anhydrosis was diagnosed based on clinical symptoms of chronic ulcers, joint deformities, malunited fractures, anhydrosis, and learning disabilities. We detected a compound heterozygous mutation in exon 16: V709L from the mother and G718S from the father. Two novel mutations were identified: at amino acid 709, a change of G to C at nucleotide 2209 (approximately 2209G to C) causing a valine to leucine substitution (V709L), and at amino acid 718, a change of G to A at nucleotide 2236 (approximately 2236G to A) causing a glycine to serine substitution (G718S). Polymorphisms identified were at nucleotides approximately 2113G to C and approximately 2176T to C.