There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn2+ on transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-beta1 (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 micromol/L Zn2+ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-beta1-treated hepatocytes. Surprisingly, Zn2+ did not inhibit the formation of high-molecular-weight DNA fragments (30-50 kbp to 250-300 kbp). These data provide evidence that Zn2+ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-beta1-induced apoptosis in hepatocytes.
Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype.
On 15 September 1995 a Malaysian Airlines (MAS) Fokker 50 plane plunged while descending and crashed, killing thirty-four passengers aboard. The dental disaster victim identification team comprising dental surgeons from the Dental faculty, University of Malaya; Ministry of Health, Sabah; and the Malaysian Defence Forces played an active role in the identification process. Most of the bodies were badly mutilated, disfigured and severely incinerated. Problems were encountered due to inadequate facilities and space at the mortuary. Difficulties were also encountered during the procurement and deciphering of information from dental records. This disaster has however created greater awareness amongst Malaysians of the important role of forensic odontology in mass disasters.
In this study, the genome of the Plasmodium falciparum Gombak A strain was examined for the presence of a gene encoding falcipain-2, a cysteine protease, using homology-based polymerase chain reaction cloning. The nucleotide sequence obtained from the gene cloned (designated pFG1) is approximately 99% homologous to other falcipain-2 genes from different strains. Comparatively, it is 69% homologous to falcipain-3 genes. Direct cloning of the falcipain-2 gene and its resemblance to the reported corresponding mRNA transcript suggests the absence of introns in this gene. Sequence alignment and comparison revealed four amino acid differences at positions 15, 51, 59 and 414 in the falcipain-2 from P. falciparum Gombak A as compared to other falcipain-2 proteins from different strains.
We have examined 56 unrelated individuals from Malaysian aborigines for their DNA polymorphism of the HLA-B gene by sequence specific oligonucleotide probe (SSO) method. Using the SSO hybridization, we found that one specific DNA allele with a B*1513 like pattern of epitope combination (ECB1513) was dominant among the Melayu Asli (Af = 41.9%) and the Senoi (Af = 24%). To determine the nucleotide sequences of ECB1513, a DNA fragment spanning from the beginning of exon 1 to the middle of exon 4 of the HLA-B gene was amplified by polymerase chain reaction (PCR) from two ECB1513 positive individuals, and the PCR products were cloned and sequenced. This sequencing analysis confirmed that ECB1513 was identical to HLA-B*1513 in exon 1, 2, 3, and 4. Amino acid sequence of this major allele, HLA-B*1513, in the aborigines especially around the peptide binding groove (B and F pockets), was compared with that of African B*5301 that had been suggested to confer resistance to malaria infection in Africa. The amino acid residues composing of the F pocket were completely identical in B*1513 and B*5301. These observations suggest that a common environmental factor, the malaria infection, might have independently enhanced the selection of functional change in the polymorphic portion of HLA-B gene in Africa and in South-East Asia.
A modified nested polymerase chain reaction (PCR) method for detection of Plasmodium falciparum, P. vivax and P. malariae was combined with a simple blood collection and deoxyribonucleic acid (DNA) extraction method and evaluated in Malaysia. Finger-prick blood samples from 46 hospital patients and 120 individuals living in malaria endemic areas were spotted on filter papers and dried. The simple Chelex method was used to prepare DNA templates for the nested PCR assay. Higher malaria prevalence rates for both clinical (78.2%) and field samples (30.8%) were obtained with the nested PCR method than by microscopy (76.1% and 27.5%, respectively). Nested PCR was more sensitive than microscopy in detecting mixed P. falciparum and P. vivax infections, detected 5 more malaria samples than microscopy on the first round of microscopical examination, and detected malaria in 3 microscopically negative samples. Nested PCR failed to detect parasite DNA in 2 microscopically positive samples, an overall sensitivity of 97.4% compared to microscopy. The nested PCR method, when coupled with simple dried blood spot sampling, is a useful tool for collecting accurate malaria epidemiological data, particularly in remote regions of the world.
The spectrum of beta-thalassemia mutations in Malays in Singapore and Kelantan (Northeast Malaysia) was studied. Allele specific priming was used to determine the mutations in beta-carriers at -28, Codon 17, IVSI #1, IVSI #5, Codon 41-42 and IVSII #654 along the beta-globin gene. The most common structural hemoglobin variant in Southeast Asia, Hb E, was detected by DNA amplification with restriction enzyme (Mnl1) analysis. Direct genomic sequencing was carried out to detect the beta-mutations uncharacterized by allele-specific priming. The most prevalent beta-mutations in Singaporean Malays were IVSI #5 (45.83%) followed by Hb E (20.83%), codon 15 (12.5%) and IVSI #1 and IVSII #654 at 4.17% each. In contrast, the distribution of the beta-mutations in Kelantan Malays differed, with Hb E as the most common mutation (39.29%) followed by IVSI #5 (17.86%), codon 41-42 (14.29%), codon 19 (10.71%) and codon 17 (3.57%). The beta-mutations in Kelantan Malays follow closely the distribution of beta-mutations in Thais and Malays of Southern Thailand and Malays of West Malaysia. The AAC-->AGC base substitution in codon 19 has been detected only in these populations. The spectrum of beta-mutations in the Singaporean Malays is more similar to those reported in Indonesia with the beta-mutation at codon 15 (TGG-->TAG) present in both populations. The characterization of beta-mutations in Singaporean and Kelantan Malays will facilitate the establishment of effective prenatal diagnosis programs for beta-thalassemia major in this ethnic group.
The blood filtration method was used as the gold standard to determine the detection level of simple blood-spot sampling and nested-polymerase chain reaction (PCR) for Brugia malayi. Of 100 samples, 48 were filtration-positive. Of these, 26 had microfilaria counts that were low enough (<1-29 microfilariae/ml) to accurately assess the limit of detection by nested-PCR. Nested-PCR consistently detected B. malayi DNA in samples with > or = 10 microfilariae/ml. Post-filtration, microfilaria-depleted, blood-spots from microfilaria-positive samples were screened by nested-PCR and B. malayi specific 'free' DNA was detected in 51.7% of these samples. There was no evidence for 'free' DNA in microfilaria-negative individuals from this endemic community.
The Japanese encephalitis (JE) serocomplex of flaviviruses comprises 10 members, 9 of which: Alfuy (ALF); Koutango (KOU); Kokobera (KOK); Kunjin (KUN); Murray Valley encephalitis (MVE); JE; Stratford (STR); Usutu (USU); and West Nile (WN) have been isolated from Africa, southern Europe, Middle East, Asia, and Australia. The tenth member, St. Louis encephalitis (SLE) virus, is confined to North, Central, and South America. For ALF, KOK, KOU, STR, and USU, no sequence data have as yet been reported, and little molecular phylogeny has been determined for this complex as a whole. Using a rapid, one-step RT-PCR and universal primers, we have amplified and sequenced a 450-600 base pair region of the virus genome encompassing the N terminus of the nonstructural protein NS5 and the 5' end of the 3' noncoding region, for several strains of all of these viruses, except USU and SLE viruses. These data, as well as published sequence data for other flaviviruses, were analyzed with the ClustalW and Phylip computer packages. The resultant phylogenetic data were consistent with some of the current flavivirus serological classification, showing a close relationship between ALF and MVE viruses and between KOK and STR viruses, but suggested that KOK and STR are distantly related to the other viruses and should perhaps be reclassified in their own serocomplex. The data also confirmed the close relationship between KUN and WN viruses and showed that an isolate of KUN virus from Sarawak may represent a "link" between these two virus species. In addition, the primary sequence data revealed a polymorphic region just downstream of the stop codon in the 3' end of the viral genomes.
Matched MeSH terms: Sequence Analysis, DNA; DNA Primers
Methicillin-resistant Staphylococcus aureus (MRSA) infection has been endemic in the University Hospital, Kuala Lumpur since the late 1970s. Fifty isolates of MRSA obtained from clinical specimens of patients with nosocomial infections associated with this organism have been studied by pulsed-field gel electrophoresis (PFGE) of its chromosomal DNA fragments to discrimate between strains and to identify the predominant strain. Twenty-one chromosomal patterns were observed which could be further grouped into nine types. The predominant strain was Type 9-b (40% of isolates) found mainly in the Orthopaedic and Surgical Units. Outbreak strains found in the Special Care Nursery were of Type 1, entirely different from those of the surgical ward S2, which were of Type 9-b. Type 8 strains were found mainly at one end of the hospital building where the maternity, paediatric and orthopaedic units were situated. Genomic DNA fingerprinting by PFGE is recommended as a useful and effective tool for the purpose of epidemiological studies of MSRA infections, particularly for nosocomial infections.
Plasmodium inui (Halberstaedter and von Prowazek, 1907), a malarial parasite of Old World monkeys that occurs in isolated pockets throughout the Celebes, Indonesia, Malaysia, and the Philippines, has traditionally been considered to be related more closely to Plasmodium malariae of humans (and its primate counterpart Plasmodium brasilianum), than to other primate Plasmodium species. This inference was made in part because of the similarities in the periodicities or duration of the asexual cycle in the blood, the extended sporogonic cycle, and the longer period of time for development of the pre-erythrocytic stages in the liver. Both P. inui and P. malariae have quartan (72 hr) periodicities associated with their asexual cycle, whereas other primate malarias, such as Plasmodium fragile and Plasmodium cynomolgi, are associated with tertian periodicities (48 hr), and Plasmodiumn knowlesi, with a quotidian (24 hr) periodicity. Phylogenetic analyses of portions of orthologous small subunit ribosomal genes reveal that P. inui is actually more closely related to the Plasmodium species of the "vivax-type" lineage than to P. malariae. Ribosomal sequence analysis of many different, geographically isolated, antigenically distinct P. inui isolates reveals that the isolates are nearly identical in sequence and thus members of the same species.
One of the causative agents of lympahtic filariasis is the nematode parasite Brugia malayi that requires a competent mosquito vector for its development and transmission. Armigeres subalbatus mosquitoes rapidly destroy invading B. malayi microfilariae via a defense response known as melanotic encapsulation. We have constructed a genetic linkage map for this mosquito species using RFLP markers from Aedes aegypti. This heterologous approach was possible because of the conserved nature of the coding sequences used as markers and provided an experimental framework to evaluate the hypothesis that linkage and gene order are conserved between these mosquito species. Of the 56 Ae. aegypti markers tested, 77% hybridize to genomic DNA digests of Ar. subalbatus under stringent conditions, with 53% of these demonstrating strain-specific polymorphisms. Twenty-six Ae. aegypti markers have been mapped using an F2- segregating Ar. subalbatus population derived from a cross of strains originating in Japan and Malaysia. Linear order of these marker loci is highly conserved between the two species. Only 1 of these markers, LF92, was not linked in the manner predicted by the Ae. aegypti map. In addition, the autosomal sex-determination locus that occurs in linkage group 1 in Ae. aegypti resides in group 3 in Ar. subalbatus. The Ar. subalbatus map provides a basic genetic context that can be utilized in further genetic studies to clarify the genetic basis of parasite resistance in this mosquito and is a necessary precursor to the identification of genome regions that carry genes that determine the encapsulation phenotype. [The composite map and sequence database information for Ae. aegypti markers can be retrieved directly from the Ae. aegypti Genome Database through the World Wide Web: http://klab.agsci.colostate.edu.]
Associations among seven apolipoprotein B (APOB) gene polymorphisms [C-T promoter site; Leu-Ala-Leu signal peptide (SP) insertion/deletion; AG C,G site at codon 71; AG A1,D site at codon 591; XbaI site at codon 2488; AG H,I site at codon 3611; and AG T,Z site at codon 4154] were investigated in 195 members of an Orang Asli (aborigine) population from western Malaysia. Frequencies of the rare alleles for all these polymorphisms turned out to be low when compared with European but not Asian populations. The AG H,I site was not polymorphic. The highly polymorphic sites are in linkage disequilibrium among themselves, as shown by their delta values: SP 24,27 and AG C,G, 0.68; SP 24,27 and AG A1,D, 0.71; XbaI and AG C,G, 0.64; XbaI and AG A1,D, 0.57; SP 24,27 and XbaI, 0.48; and AG C,G and AG A1,D, 0.68. Ten unequivocal haplotypes on the basis of six sites (excluding the promoter polymorphism) were observed, and they represent 80% of the sample. The frequency of haplotype SP27,G,A1,X-,I,T, defined by the common homozygotes at all the sites for the APOB gene was 0.7, compared with 0.22 in Europeans. The ancestral haplotype SP27,G,D,X-,I,T was present at low frequency (0.01) in both the Orang Asli and Europeans. A cladogram constructed on the basis of haplotypes in the Orang Asli shows two different lines of evolution and that other haplotypes evolved by subsequent mutations on the ancestral haplotype.
The genomic relationships of wild poliovirus type 1 strains recently isolated in Europe, the Middle East, and the Indian subcontinent was analyzed by automated amplicon sequencing of the VP1/2A junction region of the genome. Four major genotypes of poliovirus type 1 were found to circulate. Two genotypes were found predominantly in Eastern Europe, one of these in the Caucasian Region and the other in countries bordering the Black Sea. A third genotype circulated mainly in Egypt. The fourth and largest genotype circulated in the largest geographic area. Strains belonging to this genotype could be found in countries as far apart as Malaysia and Ukraine. Considerable genetic variation was observed among strains isolated in Egypt, Pakistan, and India, where poliovirus is endemic. Strains belonging to all four genotypes circulated in Pakistan. Data confirm the extent of poliovirus circulation in certain regions, stressing the need for intensification of vaccination in these regions.
The protozoan parasite Eimeria tenella has a complex life cycle that includes two major asexual developmental stages, the merozoite and the sporozoite. The expressed sequence tag (EST) approach has been previously used to study gene expression of merozoites. We report here the generation and analysis of 556 ESTs from sporozoites. Comparative analyses of the two datasets reveal a number of transcripts that are preferentially expressed in a specific stage, including previously uncharacterised sequences. The data presented indicate the invaluable potential of the comparative EST analysis for providing information on gene expression patterns in the different developmental stages of E. tenella.
BACKGROUND: Hair roots provide a useful alternative to blood as a source of DNA for genotyping. Besides simple and non-invasive collections, the DNA extraction step is also easy to perform and is fast. The aim of our study is to determine if hair roots can be used to genotype all of the common CYP2D6 alleles for routine screening purposes.
METHOD: The study complies with the Declaration of Helsinki. After obtaining informed consents, both blood and hair samples were collected from 92 patients for genotyping of the CYP2D6 gene. PCR was used to detect the following mutations: CYP2D6*1, *3, *4, *5, *9, *10, *17 and duplication gene. The results were compared where hair roots and blood were used as templates for DNA respectively.
RESULTS: When blood was used as a source of DNA for genotyping, all of the investigated CYP2D6 alleles were successfully amplified. However, with hair roots, the genes with the larger fragment sizes: CYP2D6*5 and the duplication gene could not be amplified and the bands of other alleles investigated were faint when visualized under UV light.
CONCLUSIONS: DNA extraction from hair roots and leucocytes yielded similar results but the DNA extracted from hair roots did not allow successful amplification of the longer genes such as the CYP2D6*5 and the duplication gene.
The genetic differentiation of nuclear, mitochondrial (mt) and chloroplast (cp) genomes was investigated by Southern and PCR analysis using 75 varieties of cultivated rice ( Oryza sativa L.) and 118 strains of common wild rice (CWR, Oryza rufipogon Griff.) from ten countries of Asia. The distinguishing differences between the Indica and Japonica cultivars were detected both in the nuclear genome and the cytoplasmic genome, confirming that the Indica-Japonica differentiation is of major importance for the three different classes of genome in cultivated rice. This differentiation was also detected in common wild rice with some differences among the genome compartments and the various regions. For nuclear DNA variation, both Indica-like and Japonica-like types were observed in the Chinese CWR, with the latter more-frequent than the former. No Japonica-like type was found in South Asia, and only two strains of the Japonica-like type were detected in Southeast Asia, thus the Indica-like type is the major type among South and Southeast Asian CWR. For mtDNA, only a few strains of the Japonica-like type were detected in CWR. For cpDNA, the Japonica type was predominant among the CWR strains from China, Bangladesh and Burma, while the Indica type was predominant among the CWR strains from Thailand, Malaysia, Cambodia and Sri Lanka, and both types were found in similar frequencies among the Indian CWR. Altogether, however, the degree of Indica-Japonica differentiation in common wild rice was much-less important than that in cultivated rice. Cluster analyses for nuclear and mitochondrial DNA variation revealed that some CWR strains showed large genetic distances from cultivated rice and formed clusters distinct from cultivated rice. Coincidence in the genetic differentiation between the three different classes of genome was much higher in cultivated rice than in CWR. Among the 75 cultivars, about 3/4 entries were "homoeotype" showing congruent results for nuclear, mt and cpDNA regarding the Indica-Japonica differentiation. In CWR, the proportions of homoeotypes were 5.7%, 15% and 48.8% in China, South Asia and Southeast Asia, respectively. Based on the average genetic distance among all the strains of CWR and cultivated rice for nuclear and mitochondrial genomes, the variability of the nuclear genome was found to be higher than that of the mitochondrial genome. The global pattern based on all genomes shows much-more diversification in CWR than that in cultivated rice.
To elucidate the evolution of one of the most species-rich ant-plant symbiotic systems, the association between Crematogaster (Myrmicinae) and Macaranga (Euphorbiaceae) in South-East Asia, we conducted a phylogenetic analysis of the ant partners. For the phylogenetic analysis partial mitochondrial cytochrome oxidase I and II were sequenced and Maximum Parsimony analysis was performed. The analyzed Crematogaster of the subgenus Decacrema fell into three distinct clades which are also characterized by specific morphological and ecological traits (queen morphology, host-plants, and colony structure). Our results supported the validity of our currently used morphospecies concept for Peninsula Malaysia. However, on a wider geographic range (including North and North-East Borneo) some morphospecies turned out to be species complexes with genetically quite distinct taxa. Our phylogenetic analysis and host association studies do not indicate strict cocladogenesis between the subgenus Decacrema and their Macaranga host-plants because multiple ant taxa occur on quite distinct host-plants belonging to different clades within in the genus Macaranga. These results support the view that host-shifting or host-expansion is common in the ants colonizing Macaranga. Additionally, the considerable geographic substructuring found in the phylogenetic trees of the ants suggests that allopatric speciation has also played a role in the diversification and the current distribution of the Decacrema ants.
The objective of this study was to evaluate the utility of a polymerase chain reaction (PCR) assay in detecting Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) specimens of patients suspected of having active pulmonary tuberculosis (TB) but who were sputum smear-negative. Patients undergoing investigation for suspected pulmonary TB at the University Hospital, Kuala Lumpur, and who were sputum smear-negative underwent fibreoptic bronchoscopy and BAL. One portion of each lavage specimen was submitted for smear examination for acid-fast bacilli and mycobacterial culture and the other portion assayed by PCR for the presence of a 562-base pair DNA segment belonging to the insertion sequence IS986, unique to the M. tuberculosis complex. As controls, lavage specimens from patients with other lung lesions were also similarly tested. The PCR assay gave a positivity rate of 80.9% (55 of 68) compared with 8.8% of smear examination and 7.4% of culture for detecting M. tuberculosis in BAL specimens. The assay was positive in two of 45 BAL specimens from 35 control subjects. The PCR assay was more sensitive than smear and culture in detecting M. tuberculosis in BAL specimens of patients with sputum smear-negative pulmonary TB.
OBJECTIVE: Pulsed-field gel electrophoresis (PFGE) was used to investigate an outbreak of gastroenteritis caused by Salmonella enteritidis. The outbreak occurred among university undergraduates who consumed contaminated food.
METHOD: Molecular typing was done by analyzing DNA band patterns of isolates of S. enteritidis after digestion of chromosomal DNA with infrequently-cutting restriction endonucleases XbaI, AvrII, and SpeI and separation of DNA fragments using PFGE.
RESULTS: Twenty-nine outbreak isolates of S. enteritidis had identical or highly similar PFGE patterns, whereas different PFGE patterns were observed among three epidemiologically unrelated isolates obtained during the same period.
CONCLUSION: The data obtained confirm the value of PFGE in epidemiologic investigations of outbreaks caused by S. enteritidis.