The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.
Detection of Toxoplasma gondii in blood by means of the polymerase chain reaction (PCR) may facilitate early diagnosis of toxoplasmosis in different groups of patients. We evaluated this approach in 42 patients presenting with ocular or psychotic diseases by comparing the sensitivity and specificity of PCR after heat treatment using a microwave oven with a standard genomic DNA extraction method for paired serum and whole blood samples. The presence of serum IgM and IgG antibodies against T. gondii was detected using a standard commercial enzyme-linked immunosorbent assay and enzyme immunoassay for IgG avidity test. Of 42 whole blood samples, PCR after microwave treatment was positive in 8 samples with a sensitivity of 73% and specificity of 100% compared to 11 samples positive by the extraction method. Although none of 42 sera samples was PCR positive by the extraction method, 7 specimens were positive after microwave treatment. This is the first study to use a microwave heat treatment, which is simple, rapid and a promising alternative method, in detecting small amounts of T. gondii DNA in human blood. Furthermore, irradiation of blood samples with microwaves allows incorporation of PCR into a practical tool for routine clinical assessment of patients with Toxoplasma infection.
The majority of spinal muscular atrophy (SMA) patients showed homozygous deletion or other mutations of SMN1. However, the genetic etiology of a significant number of SMA patients has not been clarified. Recently, mutation in the gene underlying cat SMA, limb expression 1 (LIX1), has been reported. Similarity in clinical and pathological features of cat and human SMA may give an insight into possible similarity of the genetic etiology.
Matched MeSH terms: Muscular Atrophy, Spinal/genetics*; RNA-Binding Proteins/genetics*; Asian Continental Ancestry Group/genetics*; Survival of Motor Neuron 1 Protein/genetics
Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.
The co-occurrence of JAK2 V617F mutation with BCR-ABL reciprocal translocation is uncommon. We report a 60-year-old man who initially presented with phenotype of polycythemia vera (PV), which evolved into chronic myeloid leukemia and back to PV once treatment with imatinib was commenced. JAK2 V617F mutation and BCR-ABL fusion transcripts were detected in the initial sample. However, JAK2 V617F alleles diminished when BCR-ABL mRNA burden increased and reappeared once the patient was commenced on imatinib. The dynamic interaction between JAK2 V617F and BCR-ABL implies that two independent clones exist with the JAK2 V617F clone only achieving clonal dominance when BCR-ABL positive clones are suppressed by imatinib.
Lancefield group C Streptococcus dysgalactiae is an emerging fish pathogen, which was first isolated in 2002 in Japan. Streptococcus dysgalactiae isolates collected from diseased fish in Japan (n=12), Taiwan (n=12), China (n=2), Malaysia (n=3), and Indonesia (n=1) were characterized using biased sinusoidal field gel electrophoresis (BSFGE), sodA gene sequence analysis, and antimicrobial susceptibility. These isolates exhibited high phenotypic homogeneity irrespective of the countries from where the strains were collected. Seventeen isolates were found to be resistant to oxytetracycline and carried the tet(M) gene, except for the strains collected in Taiwan and the PP1564 strain collected in China. The sodA gene sequence analysis revealed that 23 isolates were identical, except for one Japanese isolate (KNH07902), in which a single nucleotide differed from that of the other isolates. Based on BSFGE typing by ApaI macrorestriction, the isolates - including the Japanese, Taiwanese, and Chinese isolates - could be grouped into one main cluster at a 70% similarity level. However, the macrorestriction genotypes of some isolates were apparently distinct from those of the main cluster.
Children with Down syndrome and acute megakaryoblastic leukemia (DS-AMKL) have been shown to have increased sensitivity to cytarabine based chemotherapy. The excellent prognosis in patients with DS-AMKL may be due to mutations in the GATA1 gene leading to reduced expression of the enzyme cytidine deaminase. This leads to a decreased ability to convert cytarabine into its inactive metabolite, resulting in high intracellular concentration of this cytotoxic agent. We report two cases of DS-AMKL with GATA1 mutations who had poor outcome. These patients had high expression levels of cytidine deaminase mRNA transcripts. We speculate that other factors can affect overall outcome in patients with DS-AMKL irrespective of the presence of GATA1 mutations.
Cryptosporidium fragile sp. n. (Apicomplexa) is described from black-spined toads, Duttaphrynus melanostictus (Schneider) (Amphibia, Anura, Bufonidae) from the Malay Peninsula. The parasitized animals were directly imported from Malaysia and harboured C. fragile at the time of arrival. Oocysts were subspherical to elliptical with irregular contour in optical section, measuring 6.2 (5.5-7.0) x 5.5 (5.0-6.5) microm. Oocyst wall was smooth and colourless in light microscopy. The endogenous development of C. fragile in the stomach of black-spined toad was analysed in detail using light and electron microscopy. Cryptosporidian developmental stages were confined to the surface of gastric epithelial cells. In transmission experiments, C. fragile has not been infective for one fish species, four amphibian species, one species of reptile and SCID mice. Full length small subunit rRNA gene sequence was obtained. Phylogenetic reconstruction revealed distinct status of C. fragile within the clade of species with gastric localisation including Cryptosporidium muris Tyzzer, 1907, Cryptosporidium serpentis Levine, 1980 and Cryptosporidium andersoni Lindsay, Upton, Owens, Morgan, Mead et Blagburn, 2000. Described characteristics differentiate C. fragile from the currently recognized Cryptosporidium species. Our experience with the description of C. fragile has led us to revise the recommended criteria for an introduction of a new Cryptosporidium species name. C. fragile is the first species described and named from an amphibian host. Its prevalence of 83% (15/18) in black-spined toads within the 3 months after importation calls for strict quarantine measures and import regulation for lower vertebrates.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
Matched MeSH terms: Oryzias/genetics*; Zebrafish/genetics*; Fish Proteins/genetics*; Zebrafish Proteins/genetics
Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.
Matched MeSH terms: Actins/genetics; Collagen Type I/genetics; Collagen Type II/genetics; Aggrecans/genetics
The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
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.
The deletion (D) allele of the angiotensin-converting enzyme (ACE) gene has been studied in various populations in relation to hypertension (HTN) and type 2 diabetes mellitus (T2DM) with contradictory results. This study sought to determine the association of insertion (I)/D polymorphism of the ACE gene in hypertensive and T2DM subjects in a Malaysian population.
Matched MeSH terms: Diabetes Mellitus, Type 2/genetics; Hypertension/genetics; Peptidyl-Dipeptidase A/genetics*; Asian Continental Ancestry Group/genetics
Organophosphate (OP)-containing pesticides are widely used worldwide for domestic and industrial purposes. Studies on acute and chronic exposure to OPs have revealed numerous health effects attributed mainly to acetylcholinesterase (AChE) inhibition. The enzyme human serum paraoxonase (PON1) is involved in the detoxification of OP compounds. PON1 polymorphisms have been shown to affect susceptibility to OP exposure. We studied the effect of OP exposure on pest control workers and assessed the distribution of two common PON1 polymorphisms in our local population. The exposed group consisted of 103 workers from various pest control companies under the Singapore Pest Management Association while the 91 unexposed workers were from a lead stabilizer factory. For all workers, the mean age was 36.9 (20-70) years and the ethnic distribution was 38.1% Chinese, 44.3% Malay and 17.5% Indian. The mean+/-S.D. exposure duration among the pesticide workers was 10.4+/-8.4 years. The mean+/-S.D. RBC cholinesterase level was 18436.2+/-2078U/L and 18079.6+/-1576U/L for the exposed and unexposed groups, respectively (p=0.216). The mean+/-S.D. serum pseudocholinesterase was 11028.4+/-2867.4U/L and 9433.6+/-2022.6U/L in the exposed and unexposed groups, respectively (p<0.0001). Mean paraoxonase activity was similar among Chinese and Malays (266.5 and 266.3U/L, respectively) whereas that of the Indians was significantly lower (165.6U/L). Our study showed that cholinesterase levels among the exposed were not lower than those in the unexposed group. PON1 polymorphisms differed among ethnic groups, implying that ethnicity could be an important surrogate for identifying susceptible groups in case of OP exposure. Although OP poisoning is rare among occupationally exposed workers in Singapore, this information is useful for other developing countries that have large populations of Chinese, Malays and Indians where OP exposure could be very high especially in agricultural settings.
The continuing rise in tuberculosis incidence and the problem of drug resistance strains have prompted the research on new drug candidates and the mechanism of drug resistance. Molecular docking and molecular dynamics simulation (MD) were performed to study the binding of isoniazid onto the active site of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) in an attempt to address the mycobacterial resistance against isoniazid. Results show that isonicotinic acyl-NADH (INADH) has an extremely high binding affinity toward the wild type InhA by forming stronger interactions compared to the parent drug (isoniazid) (INH). Due to the increase of hydrophobicity and reduction in the side chain's volume of A94 of mutant type InhA, both INADH and the mutated protein become more mobile. Due to this reason, the molecular interactions of INADH with mutant type are weaker than that observed with the wild type. However, the reduced interaction caused by the fluctuation of INADH and the mutant protein only inflected minor resistance in the mutant strain as inferred from free energy calculation. MD results also showed there exists a water-mediated hydrogen bond between INADH and InhA. However, the bridged water molecule is only present in the INADH-wild type complex, reflecting the putative role of the water molecule in the binding of INADH to the wild type protein. The results support the assumption that the conversion of prodrug isoniazid into its active form INADH is mediated by KatG as a necessary step prior to target binding on InhA. Our findings also contribute to a better understanding of INH resistance in mutant type.
Nasopharyngeal carcinoma (NPC) is a human epithelial tumour with high prevalence amongst Chinese in Southern China and South East Asia and is associated with the Epstein-Barr virus (EBV). The viral genome harbours an oncogene, namely, the latent membrane protein 1 (LMP1) gene and known variants such as the 30-bp deletion and loss of XhoI restriction site have been found. Less is known about the relationship between these variants and the population characteristics and histological type.
The objective of this study was to investigate the modulatory effect of Chlorella vulgaris on cultured fibroblast cells derived from young and old aged individuals focusing on DNA damage, telomere length and telomerase activity. Dose-response test of the algal extract on cells in both age groups revealed that optimum viability was observed at a concentration of 50 microg/ml. Results obtained showed that Chlorella vulgaris exhibited protective effects against H(2)O(2)-induced oxidative stress as shown by the reduction in damaged DNA caused by H(2)O(2) treatment (p<0.05) in Chlorella vulgaris pre- and post-treated groups (p<0.05). Pre-treatment of Chlorella vulgaris resulted in a significant decrease in DNA damage suggesting a bioprotective effect against free radical attacks. A decline in DNA damage was observed in post-treated cells which proves Chlorella vulgaris to present bioremediative properties. In cells induced with oxidative stress, telomere length decreased significantly coupled with a concomitant decline of telomerase activity (p<0.05). However, these reductions were prevented with prior and post treatment of Chlorella vulgaris. Therefore, we concluded that Chlorella vulgaris exhibited bioprotective effects especially in cells obtained from young donor but were more bioremediative for cells obtained from old donor as indicated by DNA damage, telomere shortening and reduction in telomerase activity.
Matched MeSH terms: DNA Damage/genetics; Telomere/genetics; Oxidative Stress/genetics; Telomerase/genetics
Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.
Matched MeSH terms: DNA, Bacterial/genetics; DNA Primers/genetics; Salmonella enterica/genetics*; Drug Resistance, Bacterial/genetics
Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.
Choline-binding proteins (CBP) have been associated with the pathogenesis of Streptococcus pneumoniae. We screened, using PCR, for the presence of genes (cbpA, D, E, G) encoding these proteins in 34 isolates of pneumococci of known serotypes and penicillin susceptibility from invasive and non-invasive disease. All isolates harboured cbpD and cbpE whereas cbpA and cbpG were found in 47% and 59% respectively; the latter were more frequent in vaccine-associated types and together accounted for 77% of these isolates. No association was observed with penicillin susceptibility but 85% of non-invasive isolates were positive for these genes.