A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).
Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6')-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4- ≥ 32 μ g/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6')-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μ g/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6')-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.
Phytocystatin, a type of protease inhibitor (PI), plays major roles in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance. Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine. It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty. In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species. A full-length cDNA of the phytocystatin gene, designated CypCl, was acquired using 5'/3' rapid amplification of cDNA ends method and it has been deposited in NCBI database (accession number KF545954.1). It has a 687 bp long open reading frame (ORF) which encodes 228 amino acids. BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumis sativus with 74% max identity. Sequence analysis showed that CypCl contains most of the motifs found in a cystatin, including a G residue, LARFAV-, QxVxG sequence, PW dipeptide, and SNSL sequence at C-terminal extension. Phylogenetic studies also showed that CypCl is related to phytocystatin from Elaeis guineensis.
Dengue virus infection is a common tropical disease which often occurs without being detected. These asymptomatic cases provide information in relation to the manifestation of immunological aspects. In this study, we developed an ELISA method to compare neutralizing effects of dengue prM and E antibodies between dengue patients and their asymptomatic household members. Recombinant D2 premembrane (prM) was constructed, cloned, and tested for antigenicity. The recombinant protein was purified and tested with controls by using an indirect ELISA method. Positive dengue serum samples with their asymptomatic pair were then carried out onto the developed ELISA. In addition, commercially available recombinant envelope (E) protein was used to develop an ELISA which was tested with the same set of serum samples in the prM ELISA. Asymptomatic individuals showed preexisting heterotypic neutralizing antibodies. The recombinant prM was antigenically reactive in the developed ELISA. Dengue patients had higher prM and E antibodies compared to their household members. Our study highlights the neutralizing antibodies levels with respect to dengue prM and E between dengue patients and asymptomatic individuals.
Two field isolates of Rhizoctonia solani were isolated from infected paddy plants in Malaysia. These isolates were verified via ITS-rDNA analysis that yielded ~720 bp products of the ITS1-5.8S-ITS4 region, respectively. The sequenced products showed insertion and substitution incidences which may result in strain diversity and possible variation in disease severity. These strains showed some regional and host-specific relatedness via Maximum Likelihood and further phylogenetic analysis via Maximum Parsimony showed that these strains were closely related to R. solani AG1-1A (with 99-100% identity). Subsequent to strain verification and analysis, these isolates were used in the screening of twenty rice varieties for tolerance or resistance to sheath blight via mycelial plug method where both isolates (1801 and 1802) showed resistance or moderate resistance to Teqing, TETEP, and Jasmine 85. Isolate 1802 was more virulent based on the disease severity index values. This study also showed that the mycelial plug techniques were efficient in providing uniform inoculum and humidity for screening. In addition this study shows that the disease severity index is a better mode of scoring for resistance compared to lesion length. These findings will provide a solid basis for our future breeding and screening activities at the institution.
Silicon (Si) is the second most abundant element in soil after oxygen. It is not an essential element for plant growth and formation but plays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stresses. The molecular mechanism of Si absorption and accumulation may differ between plants, such as monocotyledons and dicotyledons. Silicon absorption and accumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids. The expression level of the genes responsible for Si absorption varies in different parts of plants. In this study, Si is mainly observed in the epidermal roots' cell walls of mangrove plants compared to other parts. The present work was carried out to discover further information on Si stress responsive genes in Rhizophora apiculata, using the suppression subtractive hybridization technique. To construct the cDNA library, two-month-old seedlings were exposed to 0.5, 1, and 1.5 mM SiO2 for 15 hrs and for 1 to 6 days resulting in a total of 360 high quality ESTs gained. Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene of serine-rich protein.
Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.
Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 5(') untranslated region (UTR) of hPPARalpha gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 5(')-untranslated region of human PPARalpha gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARalpha4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment -341/+34, but lower in the region -341/-1147 as compared to the fragment -341/+34, indicating the presence of an element conferring transcriptional activation between positions -341 and +34 or alternatively, the presence of transcriptional repression between positions -341 and -1147 in the promoter B of hPPARalpha.
This study investigated the association of hepatocyte nuclear factor 4 (HNF4) alpha single nucleotide polymorphisms (SNPs) with type 2 diabetes with or without metabolic syndrome in Malaysia. Nine HNF4 alpha SNPs were genotyped in 390 type 2 diabetic subjects with metabolic syndrome, 135 type 2 diabetic subjects without metabolic syndrome, and 160 control subjects. The SNPs rs4810424, rs1884613, and rs2144908 were associated with protection against type 2 diabetes without metabolic syndrome (recessive P = 0.018, OR 0.32; P = 0.004, OR 0.25; P = 0.005, OR 0.24, respectively). The 6-SNP haplotype2 CCCGTC containing the risk genotype of these SNPs was associated with higher risk for type 2 diabetes with or without metabolic syndrome (P = 0.002, OR 2.2; P = 0.004, OR 3.1). These data suggest that HNF4 alpha SNPs and haplotypes contributed to increased type 2 diabetes risk in the Malaysian population.
This study describes the organization of the repetitive pattern in the mtDNA control region of Tomistoma schlegelii. Using newly designed primers, we detected length variations of approximately 50-100 bp among individuals, and only one individual showed a heteroplasmic band. Sequencing the region after CSB III revealed two main patterns: a repeat motif and a variable number tandem repeat (VNTR) pattern. The VNTR region, with a core unit of 104 bp, consisting of four motifs and a short AT chain, is implicated in the length variation seen among individuals of Tomistoma. A conserved motif seen in a family unit indicated that the repeat pattern was stably inherited from the maternal parent to all offspring. A combination of VNTR patterns specific to different crocodilians was seen in Tomistoma, and the overall secondary structure was shown to be similar to that in Crocodylus and Gavialis.
Laboratory pedagogy is moving away from step-by-step instructions and toward inquiry-based learning, but only now developing methods for integrating inquiry-based writing (IBW) practices into the laboratory course. Based on an earlier proposal (Science 2011;332:919), we designed and implemented an IBW sequence in a university bioinformatics course. We automatically generated unique, double-blinded, biologically plausible DNA sequences for each student. After guided instruction, students investigated sequences independently and responded through IBW writing assignments. IBW assignments were structured as condensed versions of a scientific research article, and because the sequences were double blinded, they were also assessed as authentic science and evaluated on clarity and persuasiveness. We piloted the approach in a seven-day workshop (35 students) at Perdana University in Malaysia. We observed dramatically improved student engagement and indirect evidence of improved learning outcomes over a similar workshop without IBW. Based on student feedback, initial discomfort with the writing component abated in favor of an overall positive response and increasing comfort with the high demands of student writing. Similarly, encouraging results were found in a semester length undergraduate module at the National University of Singapore (155 students).
Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.
A new electrochemical biosensor is described for voltammetric detection of gene sequence related to Trichoderma harzianum. The sensor involves immobilization of a 20 base single-stranded probe (ssDNA), which is complementary to a specific gene sequence related to T. harzianum on a gold electrode through specific adsorption. The DNA probe was used to determine the amount of target gene in solution using methylene blue (MB) as the electrochemical indicator. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the electrode. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE and the probe-modified AuE which localized to the affinity of MB. Control experiments with the non-complementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. DNA biosensor also able to detect microorganism at the species levels without nucleic acid amplification. The redox current was linearly related to the concentration of target oligonucleotide DNA, ranged from 1-20 ppm. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time.
Drug addiction is a multifactorial disorder. Researchers have posited that an individual's inherited behavioral propensity or temperament contributes to the disorder by shaping a personality strongly linked with the risk of drug abuse. Further, they hypothesize that the polymorphism of dopamine D2 receptor increases the susceptibility to and severity of addiction. We, therefore, investigated possible associations between dopamine D2 receptor (DRD2) and personality traits among intravenous heroin addicts.
Biogeographic connections between Australia and other continents are still poorly understood although the plate tectonics of the Indo-Pacific region is now well described. Eupetes macrocerus is an enigmatic taxon distributed in a small area on the Malay Peninsula and on Sumatra and Borneo. It has generally been associated with Ptilorrhoa in New Guinea on the other side of Wallace's Line, but a relationship with the West African Picathartes has also been suggested. Using three nuclear markers, we demonstrate that Eupetes is the sister taxon of the South African genus Chaetops, and their sister taxon in turn being Picathartes, with a divergence in the Eocene. Thus, this clade is distributed in remote corners of Africa and Asia, which makes the biogeographic history of these birds very intriguing. The most parsimonious explanation would be that they represent a relictual basal group in the Passerida clade established after a long-distance dispersal from the Australo-Papuan region to Africa. Many earlier taxonomic arrangements may have been based on assumptions about relationships with similar-looking forms in the same, or adjacent, biogeographic regions, and revisions with molecular data may uncover such cases of neglect of ancient relictual patterns reflecting past connections between the continents.
Protein function depends greatly on its structure. Based on this principle, it is vital to study the
protein structure in order to understand its function. This study attempts to build the predicted
model of lipase gene in Rhodococcus sp. NAM81 using homology modelling method. The
predicted structure was then used to investigate the function of protein through several
bioinformatic tools. The DNA sequence of lipase gene was obtained from the Rhodococcus sp.
NAM81 genome scaffold. Blastx analysis showed 100% identity to the target enzyme andthe
appropriate template for homology modelling was determined using Blastp analysis. The 3D
protein structure was built using two homology modelling software, EsyPred3D and Swiss
Model Server. Both structures built obtained LGScore of greater than 4, which means they are
extremely good models according to ProQ validation criteria. Both structures also satisfied the
Ramachandran plot structure validation analysis. The predicted structures were 100% matched
with each other when superimposed with DaliLite pairwise. This shows that both structure
validation servers agreed on the same model. Structure analysis using ProFunc had found seven
motifs and active sites that indicate similar function of this protein with other known proteins.
Thus, this study has successfully produced a good 3D protein structure for the target enzyme.
This study determined the most effective microbes acting as ammonia-oxidising (AOB) and manganese-oxidising bacteria (MnOB) for the simultaneous removal of ammonia (NH(4)(+)-N) and manganese (Mn(2+)) from water. Two conditions of mixed culture of bacteria: an acclimatised mixed culture (mixed culture: MC) in a 5-L bioreactor and biofilm attached on a plastic medium (stages of mixed culture: SMC) in a biological aerated filter were isolated and identified using Biolog MicroSystem and 16S rRNA sequencing. A screening test for determining the most effective microbe in the removal of NH(4)(+)-N and Mn(2+) was initially performed using SMC and MC, respectively, and found that Bacillus cereus was the most effective microbe for the removal of NH(4)(+)-N and Mn(2+). Moreover, the simultaneous NH(4)(+)-N and Mn(2+) removal (above 95% removal for both NH(4)(+)-N and Mn(2+)) was achieved using a biological aerated filter under various operating conditions. Thus, the strain could act as an effective microbe of AOB and a MnOB for the simultaneous removal of NH(4)(+)-N and Mn(2+).
This study was undertaken to investigate the effects of different nitrate concentrations in culture medium on oil content and fatty acid composition of Chlorella vulgaris (UMT-M1) and Chlorella sorokiniana (KS-MB2). Results showed that both species produced significant higher (p<0.05) oil content at nitrate ranging from 0.18 to 0.66 mM with C. vulgaris produced 10.20-11.34% dw, while C. sorokiniana produced 15.44-17.32% dw. The major fatty acids detected include C16:0, C18:0, C18:1, C18:2 and C18:3. It is interesting to note that both species displayed differentially regulated fatty acid accumulation patterns in response to nitrate treatments at early stationary growth phase. Their potential use for biodiesel application could be enhanced by exploring the concept of binary blending of the two microalgae oils using developed mathematical equations to calculate the oil mass blending ratio and simultaneously estimated the weight percentage (wt.%) of desirable fatty acid compositions.
The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.