We have defined DNA repeat variability in the 3'-terminus of the cagA gene of Helicobacter pylori strains from Malaysian patients of different ethnicities. We identified different alleles based on the EPIYA repeats. cagA types A-B-D and A-B-B-D are more similar to the sequence of Japanese strains, whereas cagA types A-B-C, A-B-C-C, A-B and A-C displayed similarity to strain 26695 sequences. A significant association was found between cagA genotypes and patients' ethnicity, with cagA type A-B-D being predominantly isolated from Chinese patients and cagA type A-B-C from Malays and Indians. Our data further corroborate the possibility that variant biological activity of CagA may affect the host specificity and/or pathogenicity of H. pylori.
A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.
P-glycoprotein (PgP) is the most extensively studied ATP-binding cassette (ABC) coded by MDR1 gene. To date, 29 single nucleotide polymorphisms (SNPs) have been identified; but only SNP C3435T has been correlated with intestinal PgP expression levels and shown to influence the absorption of orally taken drugs that are PgP substrates. Individuals homozygous for the T allele have more than fourfold lower PgP expression compared with C/C individuals. We developed a one step primer based allele specific PCR method to detect SNP at C3435T to investigate the distribution of this genotype in the local population.
The present study was done to determine the molecular epidemiology of rabies virus (RV) in Vietnam. The nucleoprotein (N) and glycoprotein (G) genes of RVs were amplified from the brains of ten rabid dogs of Ho Chi Minh City, Vietnam. The nucleotide sequences of these genes were compared with those of other Asian strains to find the possible relationship among them. Phylogenetic analysis revealed that the Asian N gene segregated into three main branches, namely South-East Asia 1 (SEA 1), South-East Asia 2 (SEA 2) and Indian subcontinent (ISC) genotypes. The SEA 1 genotype comprised RVs from Malaysia, Vietnam and Thailand. The SEA 2 genotype contained strains from the Philippines, and the ISC genotype comprised strains from Sri Lanka and India. Phylogenetically G genes of RVs from Vietnam and Thailand were clustered together. Our study suggests that Vietnamese and Thai RVs are closely related and might have originated from a common ancestor.
Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003-04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.
Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.
The MDR1 multidrug transporter represents one of the better characterized drug transporters that play an important role in protecting the body against xenobiotic insults. Single nucleotide polymorphisms (SNPs) and SNP haplotypes within this gene have been variously associated with differences in MDR1 expression/function, drug response as well as disease susceptibility. Nonetheless, the effect of polymorphisms at the MDR1 promoter region on its promoter activity remains less characterized. Through the examination of approximately 1.5 kilobases of MDR1 promoter region from five populations, including the Chinese, Malays, Indians, European Americans, and African Americans, we identified eight low-frequency SNPs, of which only two were polymorphic in at least four of the five populations examined. The other SNPs are mainly population-specific, the majority of which occur only in the African-American population. Recapitulation of the various combinations of SNP haplotypes in vitro in promoter-reporter assays revealed a few notable trends. The African and European American-specific haplotypes tended to result in enhanced MDR1 promoter activity only in the human embryonic kidney (HEK) 293 cell line. Haplotype GCTAACC, which occurs at variable frequencies in all the populations examined, with Asians having much lower frequencies (<2%) compared with the European Americans/African Americans (>4%), affected MDR1 promoter activity differently in different cell lines. Compared with the commonest haplotype, GCTA-ACC haplotype resulted in a significant decrease in MDR1 promoter activity in HeLa cells (P < 0.05) but a significant increase in the same promoter activity in HEK293 cells (P < 0.05). These results suggest that the MDR1 promoter region is largely invariant but that different haplotypes have differential effects on the MDR1 promoter activity in different cell lines.
Strategies to resolve B*18 alleles which carry a deletion in intron 1 close to the 5' end of exon 2 relative to other HLA-B alleles or a null allele mutation in exon 1 and to resolve ambiguities among allele combinations including B*18 are described. B*18 allele frequencies from volunteer donors recruited for two hematopoietic stem cell registries show the presence of two alleles, B*180101 and B*1802, in a population from Singapore and only B*180101 in African-Americans.
We performed DNA analysis on cord blood samples of 128 Chinese male neonates diagnosed as G6PD deficiency in Hospital Universiti Kebangsaan Malaysia by a combination PCR-restriction enzyme digest technique, Single Stranded Conformation Polymorphism analysis and DNA sequencing. We found 10 different G6PD-deficient mutations exist. The two commonest alleles were G6PD Canton 1376 G>T (42.3%) and Kaiping 1388 G>A (39.4%) followed by G6PD Gaohe 592 G>A (7.0%), Chinese-5 1024 C>T, Nankang 517 T>C (1.5%), Mahidol 487 G>A (1.6%), Chatham 1003 G>T (0.8%), Union 1360 C>T (0.8%), Viangchan 871 G>A (0.8%) and Quing Yang 392 G>T (0.8%). Sixty eight percent (88/125) neonates in this study had neonatal jaundice and 29.7% developed hyperbilirubinemia >250 micromol/l. The incidence of hyperbilirubinemia >250 micromol/l was higher in G6PD Kaiping (43.8%) than G6PD Canton (22%) (p< 0.05). There was no significant difference in the incidence of neonatal jaundice, mean serum bilirubin, mean age for peak serum bilirubin, percentage of babies requiring phototherapy and mean duration of phototherapy between the two major variants. None of the 88 neonates required exchange transfusion. In conclusion we have completely characterized the molecular defects of a group of Chinese G6PD deficiency in Malaysia. The mutation distribution reflects the original genetic pool and limited ethnic admixture with indigenous Malays.
Molecular diversity of protists from bovine rumen fluid incubated with condensed tannins of Leucaena leucocephala hybrid-Rendang at 20 mg/500 mg dry matter (treatment) or without condensed tannins (control) was investigated using 18S rRNA gene library. Clones from the control library were distributed within nine genera, but clones from the condensed tannin treatment clone library were related to only six genera. Diversity estimators such as abundance-based coverage estimation and Chao1 showed significant differences between the two libraries, although no differences were found based on Shannon-Weaver index and Libshuff.
An outbreak of acute hemorrhagic conjunctivitis occurred in French Guiana between April and July 2003, with approximately 6,000 cases in the two major cities Kourou and Cayenne. Since acute hemorrhagic conjunctivitis is not a notifiable disease in France, there was no registration of the number of cases. Therefore, these were estimated by comparing the consumption of antibiotic eye drops and ophthalmic ointments during 2002 and 2003. The outbreak rapidly spread into the Caribbean Islands, causing an outbreak in Guadeloupe in October. Viral isolates from conjunctival swabs of 16 patients were confirmed to be enterovirus by PCR directed to the 5' UTR of the genome. The isolates could not be neutralized by the Melnick intersecting pools, but were shown to be CV-A24 variant by limited sequencing within the VP1 and 3C regions of 12 strains. Phylogenetic analysis revealed that they were similar to the genotype III strains causing outbreaks in Korea 2002 and Malaysia 2003. The previous outbreak of conjunctivitis caused by CV-A24 in the Caribbean in the 1980s was also introduced from Asia, and disappeared after 3 years. This new introduction from Asia and its rapid spread into the Caribbean, where the infection disappeared after a few months, indicates that the CV-A24 variant has a different epidemiological pattern in this region compared to South East Asia, since it has not established an endemic infection. It had to be reintroduced from Asia, where it has been circulating since the 1970s.
A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.
An epidemiological study of Ehrlichia canis infection in dogs in Peninsular Malaysia was carried out using molecular detection techniques. A total of 500 canine blood samples were collected from veterinary clinics and dog shelters. Molecular screening by polymerase chain reaction (PCR) was performed using genus-specific primers followed by PCR using E. canis species-specific primers. Ten out of 500 dogs were positive for E. canis. A phylogenetic analysis of the E. canis Malaysia strain showed that it was grouped tightly with other E. canis strains from different geographic regions. The present study revealed for the first time, the presence of genetically confirmed E. canis with a prevalence rate of 2.0% in naturally infected dogs in Malaysia.
Commercial fisheries of lumpfish Cyclopterus lumpus have been carried out in Iceland for centuries. Traditionally the most valuable part is the eggs which are harvested for use as a caviar substitute.Previously reported parasitic infections from lumpfish include an undescribed intranuclear microsporidian associated with abnormal kidneys and mortalities in captive lumpfish in Canada. During Icelandic lumpfish fisheries in spring 2011, extensive enlargements to the kidneys were observed in some fish during processing. The aim of this study was to identify the pathogen responsible for these abnormalities.
Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.
alpha-Galactosidase (EC 3.2.1.22) from ripe papaya (Carica papaya L.) fruit was fractionated by a combination of ion exchange and gel filtration chromatography into three forms, viz., alpha-galactosidase 1, 2 and 3. The predominant isoform, alpha-gal 2, was probably a tetramer with a native molecular mass of about 170 kDa and 52 kDa-sized subunits and an estimated pI of 7.3. The subunit's N-terminal amino acid sequence shared high identity (97%) with the deduced sequence of a papaya cDNA clone encoding a putative alpha-galactosidase PAG2 as well as with an Ajuga reptans L. GGT1 clone encoding a galactan: galactan galactosyltransferase (66%). During ripening, alpha-galactosidase activity increased concomitantly with firmness loss and this increase was largely ascribed to alpha-gal 2. The protein level of alpha-gal 2 as estimated by immunoblot was low in developing fruits and generally increased with ripening. alpha-Galactosidase 2 also had the ability to markedly catalyse increased pectin solubility and depolymerisation while the polymers were still structurally attached to the cell walls mimicking, in part, the changes that occur during ripening. The close correlation between texture changes, alpha-gal 2 activity and protein levels as well as capability to modify intact cell walls suggest that the enzyme might contribute to papaya fruit softening during ripening. The purported mechanism of alpha-gal 2 action as a softening enzyme was discussed in terms of its functional capacity as a glycanase or perhaps, as a transglycosylase.
Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.
At least three different EV-71 subgenotypes were identified from an outbreak in Malaysia in 1998. The subgenotypes C2 and B4 were associated with the severe and fatal infections, whereas the B3 virus was associated with mild to subclinical infections. The B3 virus genome sequences had >= 85% similarity at the 3' end to CV-A16. This offers opportunities to examine if there are characteristic similarities and differences in virulence between CV-A16, EV-71 B3 and EV-71 B4 and to determine if the presence of the CV-A16-liked genes in EV-71 B3 would also confer the virus with a CV-A16-liked neurovirulence in mice model infection.
ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.