Displaying publications 741 - 760 of 1608 in total

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  1. Induction of protein expression within Escherichia coli vector for entry into mammalian cells
    Chen Q, Lee CW, Sim EU, Narayanan K
    Hum Gene Ther Methods, 2014 Feb;25(1):40-7.
    PMID: 24134118 DOI: 10.1089/hgtb.2012.188
    Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.
    Matched MeSH terms: Viral Proteins/genetics*
  2. Fulltext The complete genome sequence of EC1-UPM, a novel N4-like bacteriophage that infects Escherichia coli O78:K80
    Gan HM, Sieo CC, Tang SG, Omar AR, Ho YW
    Virol J, 2013;10:308.
    PMID: 24134834 DOI: 10.1186/1743-422X-10-308
    Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
    Matched MeSH terms: Viral Proteins/genetics
  3. Phenotypic and genotypic characterization of induced acyclovir-resistant clinical isolates of herpes simplex virus type 1
    Hussin A, Md Nor NS, Ibrahim N
    Antiviral Res, 2013 Nov;100(2):306-13.
    PMID: 24055837 DOI: 10.1016/j.antiviral.2013.09.008
    Eleven strains of acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) were generated from HSV-1 clinical isolates by exposure to ACV. Genotype of the thymidine kinase (TK) and DNA polymerase (pol) genes from these mutants were further analyzed. Genotypic analysis revealed four non-synonymous mutations in TK gene associated with gene polymorphism and two to three non-synonymous mutations in DNA pol gene. Seven and six strains contained at least one resistance-associated mutation at TK and DNA pol gene, respectively. Resistance-associated mutations within the TK gene consisted of 64% of non-synonymous frameshift mutations within the homopolymer region of G's and C's, and 36% of non-synonymous nucleotide substitutions of the conserved gene region (C336Y, R51W and R222H), nucleotide that produced stop codon (L288Stop) and two amino acid substitutions outside the conserved region (E39G & L208F). There were 10 non-synonymous amino acid substitutions located outside the conserved region with the unclear significance to confer resistance observed. Resistance-associated mutations in DNA pol gene include insertion of G at the homopolymer region of G's (794-797) and amino acid substitutions inside (V621S) or outside (H1228D) the conserved region. In silico analysis of the mutated TK (C336Y, R51W and L208F), and DNA pol (V621S and H1228D) suggested structural changes that might alter the stability of these proteins. However, there were several mutations with unclear significance to confer ACV-resistance identified, especially mutations outside the conserved region.
    Matched MeSH terms: Viral Proteins/genetics
  4. Cloning, expression, and purification of the hemolysin/cytolysin (HlyE antigen) from Salmonella enterica serovar Typhi: potential application for immunoassay development
    Ong EB, Anthony AA, Ismail A, Ismail A, Lim TS
    Diagn Microbiol Infect Dis, 2013 Sep;77(1):87-9.
    PMID: 23790417 DOI: 10.1016/j.diagmicrobio.2013.05.010
    The hemolysin (HlyE) protein of Salmonella enterica serovar Typhi was reported to be antigenic. This work describes the cloning, expression, and purification of a hexahistidine-tagged HlyE protein under native conditions. Immunoblot analysis and a competitive enzyme-linked immunosorbent assay using sera from typhoid patients showed the presence of HlyE-specific antibodies in circulation.
    Matched MeSH terms: Recombinant Proteins/genetics
  5. Fulltext Evaluation of recombinant Plasmodium knowlesi merozoite surface protein-1(33) for detection of human malaria
    Cheong FW, Lau YL, Fong MY, Mahmud R
    Am J Trop Med Hyg, 2013 May;88(5):835-40.
    PMID: 23509118 DOI: 10.4269/ajtmh.12-0250
    Plasmodium knowlesi is now known as the fifth Plasmodium species that can cause human malaria. The Plasmodium merozoite surface protein (MSP) has been reported to be potential target for vaccination and diagnosis of malaria. MSP-1(33) has been shown to be immunogenic and its T cell epitopes could mediate cellular immune protection. However, limited studies have focused on P. knowlesi MSP-133. In this study, an approximately 28-kDa recombinant P. knowlesi MSP-1(33) (pkMSP-1(33)) was expressed by using an Escherichia coli system. The purified pkMSP-1(33) reacted with serum samples of patients infected with P. knowlesi (31 of 31, 100%) and non-P. knowlesi malaria (27 of 28, 96.43%) by Western blotting. The pkMSP-1(33) also reacted with P. knowlesi (25 of 31, 80.65%) and non-P. knowlesi malaria sera (20 of 28, 71.43%) in an enzyme-linked immunosorbent assay (ELISA). Most of the non-malarial infection (49 of 52 in by Western blotting and 46 of 52 in the ELISA) and healthy donor serum samples (65 of 65 by Western blotting and ELISA) did not react with recombinant pkMSP-1(33).
    Matched MeSH terms: Recombinant Proteins/genetics
  6. Fulltext In-depth tanscriptomic analysis on giant freshwater prawns
    Mohd-Shamsudin MI, Kang Y, Lili Z, Tan TT, Kwong QB, Liu H, et al.
    PLoS One, 2013;8(5):e60839.
    PMID: 23734171 DOI: 10.1371/journal.pone.0060839
    Gene discovery in the Malaysian giant freshwater prawn (Macrobrachium rosenbergii) has been limited to small scale data collection, despite great interest in various research fields related to the commercial significance of this species. Next generation sequencing technologies that have been developed recently and enabled whole transcriptome sequencing (RNA-seq), have allowed generation of large scale functional genomics data sets in a shorter time than was previously possible. Using this technology, transcriptome sequencing of three tissue types: hepatopancreas, gill and muscle, has been undertaken to generate functional genomics data for M. rosenbergii at a massive scale. De novo assembly of 75-bp paired end Ilumina reads has generated 102,230 unigenes. Sequence homology search and in silico prediction have identified known and novel protein coding candidate genes (∼24%), non-coding RNA, and repetitive elements in the transcriptome. Potential markers consisting of simple sequence repeats associated with known protein coding genes have been successfully identified. Using KEGG pathway enrichment, differentially expressed genes in different tissues were systematically represented. The functions of gill and hepatopancreas in the context of neuroactive regulation, metabolism, reproduction, environmental stress and disease responses are described and support relevant experimental studies conducted previously in M. rosenbergii and other crustaceans. This large scale gene discovery represents the most extensive transcriptome data for freshwater prawn. Comparison with model organisms has paved the path to address the possible conserved biological entities shared between vertebrates and crustaceans. The functional genomics resources generated from this study provide the basis for constructing hypotheses for future molecular research in the freshwater shrimp.
    Matched MeSH terms: Arthropod Proteins/genetics
  7. Fulltext Linkage disequilibrium between polymorphisms of ABCB1 and ABCC2 to predict the treatment outcome of Malaysians with complex partial seizures on treatment with carbamazepine mono-therapy at the Kuala Lumpur Hospital
    Subenthiran S, Abdullah NR, Joseph JP, Muniandy PK, Mok BT, Kee CC, et al.
    PLoS One, 2013;8(5):e64827.
    PMID: 23717663 DOI: 10.1371/journal.pone.0064827
    Carbamazepine (CBZ) is used as the first line of treatment of Complex Partial Seizures (CPS) in the Epilepsy Clinic, Neurology Department of Kuala Lumpur Hospital (KLH). More than 30% of the patients remain drug resistant to CBZ mono-therapy. CBZ is transported by the P-glycoprotein (P-gp). The P-gp encoded by the ABCB1 and ABCC2 genes are expressed in drug resistant patients with epilepsy. A few studies have shown significant association between CBZ resistant epilepsy and Linkage Disequilibrium (LD) with adjacent polymorphisms of these genes. Our study is aimed at determining the correlation between patients' response to CBZ mono-therapy to Single Nucleotide Polymorphisms G2677T and C3435T of the ABCB1 gene as well as G1249A and -24C>T of the ABCC2 gene.
    Matched MeSH terms: Multidrug Resistance-Associated Proteins/genetics*
  8. Fulltext Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis
    Puah SM, Puthucheary SD, Chua KH
    Int J Med Sci, 2013;10(5):539-47.
    PMID: 23532805 DOI: 10.7150/ijms.5516
    The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.
    Matched MeSH terms: Bacterial Proteins/genetics
  9. Fulltext Inhibition of dengue virus entry into target cells using synthetic antiviral peptides
    Alhoot MA, Rathinam AK, Wang SM, Manikam R, Sekaran SD
    Int J Med Sci, 2013;10(6):719-29.
    PMID: 23630436 DOI: 10.7150/ijms.5037
    Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.
    Matched MeSH terms: Viral Envelope Proteins/genetics
  10. Contribution of the MLH1 -93G>a promoter polymorphism in modulating susceptibility risk in Malaysian colorectal cancer patients
    Nizam ZM, Abdul Aziz AA, Kaur G, Abu Hassan MR, Mohd Sidek AS, Yeh LY, et al.
    Asian Pac J Cancer Prev, 2013;14(2):619-24.
    PMID: 23621208
    BACKGROUND: Colorectal cancer (CRC) exists in a more common sporadic form and less common hereditary forms, associated with the Lynch syndrome, familial adenomatous polyposis (FAP) and other rare syndromes. Sporadic CRC is believed to arise as a result of close interaction between environmental factors, including dietary and lifestyle habits, and genetic predisposition factors. In contrast, hereditary forms such as those related to the Lynch syndrome result from inheritance of germline mutations of mismatch repair (MMR) genes. However, in certain cases, the influence of low penetrance alleles in familial colorectal cancer susceptibility is also undeniable.

    AIM: To investigate the genotype frequencies of MLH1 promoter polymorphism -93G>A and to determine whether it could play any role in modulating familial and sporadic CRC susceptibility risk.

    METHODS: A case-control study comprising of 104 histopathologically confirmed CRC patients as cases (52 sporadic CRC and 52 Lynch syndrome patients) and 104 normal healthy individuals as controls was undertaken. DNA was extracted from peripheral blood and the polymorphism was genotyped employing PCR-RFLP methods. The genotypes were categorized into homozygous wild type, heterozygous and homozygous variants. The risk association between these polymorphisms and CRC susceptibility risk was calculated using binary logistic regression analysis and deriving odds ratios (ORs).

    RESULTS: When risk association was investigated for all CRC patients as a single group, the heterozygous (G/A) genotype showed a significantly higher risk for CRC susceptibility with an OR of 2.273, (95%CI: 1.133-4.558 and p-value=0.021). When analyzed specifically for the 2 types of CRC, the heterozygous (G/A) genotype showed significantly higher risk for sporadic CRC susceptibility with and OR of 3.714, (95%CI: 1.416-9.740 and p-value=0.008). Despite high OR value was observed for Lynch syndrome (OR: 1.600, 95%CI: 0.715-3.581), the risk was not statistically significant (P=0.253).

    CONCLUSION: Our results suggest an influence of MLH1 promoter polymorphism -93G>A in modulating susceptibility risk in Malaysian CRC patients, especially those with sporadic disease.

    Matched MeSH terms: Nuclear Proteins/genetics*
  11. Fulltext Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp
    Anbazhagan D, Mansor M, Yan GO, Md Yusof MY, Hassan H, Sekaran SD
    PLoS One, 2012;7(7):e36696.
    PMID: 22815678 DOI: 10.1371/journal.pone.0036696
    Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.
    Matched MeSH terms: Bacterial Proteins/genetics*
  12. Temporal changes in the genotypes of methicillin-resistant Staphylococcus aureus strains isolated from a tertiary Malaysian hospital based on MLST, spa, and mec-associated dru typing
    Lim KT, Hanifah YA, Yusof MY, Goering RV, Thong KL
    Diagn Microbiol Infect Dis, 2012 Oct;74(2):106-12.
    PMID: 22770652 DOI: 10.1016/j.diagmicrobio.2012.05.033
    Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main bacterial pathogens responsible for nosocomial infections leading to pneumonia, bloodstream, skin, and soft tissue infections. The objective of this study was to investigate the genomic changes of MRSA in a tertiary hospital between the years 2003, 2004, 2007, and 2008. One hundred fifty-four MRSA strains were characterized by multilocus sequence typing (MLST), spa, and mec-associated dru typing. Among the 154 strains, 29 different dru, 15 spa, and 8 MLST types were identified. Seven sequence types (STs) (ST239, ST22, ST5, ST6, ST80, ST573, and ST241) were identified among 2007-08 strains, although only 2 STs (ST239 and ST20) were observed among 2003 strains. Clones ST239-t037-dt13g, ST22-t032-(dt10a and dt10aw), and 28 other MRSA clones being introduced in 2007-2008 have replaced the ST239-t037 (dt13d, 14h, 13i, 13l, 13m, 15m, 15l, and 11al) clones present in 2003. The predominant MLST clone, ST239 (90.3%), was further distinguished into 7 different spa types and 26 different dru types, including 17 novel dru types. Maximum parsimony tree based on dru repeats revealed that 10 dru types (dt11am, dt13j, dt15n, dt13q, dt13n, dt13p, dt13f, dt13ao, dt12j, dt7v) shared the same MLST-spa types with dt13d, suggesting that these MRSA clones might have evolved from ST239-t037-dt13d. In conclusion, our data showed that the ST239-t037-dt13d clone and other MRSA clones in 2003 were replaced by ST239-t037-dt13g and other new emerging spa and dru types.
    Matched MeSH terms: Bacterial Proteins/genetics
  13. Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression
    Ngai SC, Rosli R, Nordin N, Veerakumarasivam A, Abdullah S
    Gene, 2012 May 1;498(2):231-6.
    PMID: 22366305 DOI: 10.1016/j.gene.2012.01.071
    Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.
    Matched MeSH terms: Green Fluorescent Proteins/genetics
  14. Fulltext Skin color variation in Orang Asli tribes of Peninsular Malaysia
    Ang KC, Ngu MS, Reid KP, Teh MS, Aida ZS, Koh DX, et al.
    PLoS One, 2012;7(8):e42752.
    PMID: 22912732 DOI: 10.1371/journal.pone.0042752
    Pigmentation is a readily scorable and quantitative human phenotype, making it an excellent model for studying multifactorial traits and diseases. Convergent human evolution from the ancestral state, darker skin, towards lighter skin colors involved divergent genetic mechanisms in people of European vs. East Asian ancestry. It is striking that the European mechanisms result in a 10-20-fold increase in skin cancer susceptibility while the East Asian mechanisms do not. Towards the mapping of genes that contribute to East Asian pigmentation there is need for one or more populations that are admixed for ancestral and East Asian ancestry, but with minimal European contribution. This requirement is fulfilled by the Senoi, one of three indigenous tribes of Peninsular Malaysia collectively known as the Orang Asli. The Senoi are thought to be an admixture of the Negrito, an ancestral dark-skinned population representing the second of three Orang Asli tribes, and regional Mongoloid populations of Indo-China such as the Proto-Malay, the third Orang Asli tribe. We have calculated skin reflectance-based melanin indices in 492 Orang Asli, which ranged from 28 (lightest) to 75 (darkest); both extremes were represented in the Senoi. Population averages were 56 for Negrito, 42 for Proto-Malay, and 46 for Senoi. The derived allele frequencies for SLC24A5 and SLC45A2 in the Senoi were 0.04 and 0.02, respectively, consistent with greater South Asian than European admixture. Females and individuals with the A111T mutation had significantly lighter skin (p = 0.001 and 0.0039, respectively). Individuals with these derived alleles were found across the spectrum of skin color, indicating an overriding effect of strong skin lightening alleles of East Asian origin. These results suggest that the Senoi are suitable for mapping East Asian skin color genes.
    Matched MeSH terms: Membrane Transport Proteins/genetics
  15. Genetic association of SNPs in the FTO gene and predisposition to obesity in Malaysian Malays
    Apalasamy YD, Ming MF, Rampal S, Bulgiba A, Mohamed Z
    Braz. J. Med. Biol. Res., 2012 Dec;45(12):1119-26.
    PMID: 22911346
    The common variants in the fat mass- and obesity-associated (FTO) gene have been previously found to be associated with obesity in various adult populations. The objective of the present study was to investigate whether the single nucleotide polymorphisms (SNPs) and linkage disequilibrium (LD) blocks in various regions of the FTO gene are associated with predisposition to obesity in Malaysian Malays. Thirty-one FTO SNPs were genotyped in 587 (158 obese and 429 non-obese) Malaysian Malay subjects. Obesity traits and lipid profiles were measured and single-marker association testing, LD testing, and haplotype association analysis were performed. LD analysis of the FTO SNPs revealed the presence of 57 regions with complete LD (D' = 1.0). In addition, we detected the association of rs17817288 with low-density lipoprotein cholesterol. The FTO gene may therefore be involved in lipid metabolism in Malaysian Malays. Two haplotype blocks were present in this region of the FTO gene, but no particular haplotype was found to be significantly associated with an increased risk of obesity in Malaysian Malays.
    Matched MeSH terms: Proteins/genetics*
  16. Fulltext Burkholderia pseudomallei transcriptional adaptation in macrophages
    Chieng S, Carreto L, Nathan S
    BMC Genomics, 2012;13:328.
    PMID: 22823543 DOI: 10.1186/1471-2164-13-328
    Burkholderia pseudomallei is a facultative intracellular pathogen of phagocytic and non-phagocytic cells. How the bacterium interacts with host macrophage cells is still not well understood and is critical to appreciate the strategies used by this bacterium to survive and how intracellular survival leads to disease manifestation.
    Matched MeSH terms: Bacterial Proteins/genetics
  17. Fulltext Simple and rapid molecular techniques for identification of amylose levels in rice varieties
    Cheng A, Ismail I, Osman M, Hashim H
    Int J Mol Sci, 2012;13(5):6156-66.
    PMID: 22754356 DOI: 10.3390/ijms13056156
    The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)(10), (CT)(11), (CT)(16), (CT)(17), and (CT)(18) were identified. Of these, (CT)(11) and (CT)(17) were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)(10) and (CT)(11), while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5'-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE.
    Matched MeSH terms: Plant Proteins/genetics*
  18. Fulltext Structural modeling and biochemical characterization of recombinant KPN_02809, a zinc-dependent metalloprotease from Klebsiella pneumoniae MGH 78578
    Wong MT, Choi SB, Kuan CS, Chua SL, Chang CH, Normi YM, et al.
    Int J Mol Sci, 2012;13(1):901-17.
    PMID: 22312293 DOI: 10.3390/ijms13010901
    Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity.
    Matched MeSH terms: Recombinant Proteins/genetics
  19. Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12
    Hashim NH, Bharudin I, Nguong DL, Higa S, Bakar FD, Nathan S, et al.
    Extremophiles, 2013 Jan;17(1):63-73.
    PMID: 23132550 DOI: 10.1007/s00792-012-0494-4
    The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.
    Matched MeSH terms: Recombinant Proteins/genetics
  20. Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris
    Cha TS, Yee W, Aziz A
    World J Microbiol Biotechnol, 2012 Apr;28(4):1771-9.
    PMID: 22805959 DOI: 10.1007/s11274-011-0991-0
    The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.
    Matched MeSH terms: Green Fluorescent Proteins/genetics
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