Displaying publications 101 - 120 of 4045 in total

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  1. Fulltext ATG16L1 rs2241880/T300A increases susceptibility to perianal Crohn's disease: An updated meta-analysis on inflammatory bowel disease risk and clinical outcomes
    Simovic I, Hilmi I, Ng RT, Chew KS, Wong SY, Lee WS, et al.
    United European Gastroenterol J, 2024 Feb;12(1):103-121.
    PMID: 37837511 DOI: 10.1002/ueg2.12477
    BACKGROUND: ATG16L1 plays a fundamental role in the degradative intracellular pathway known as autophagy, being a mediator of inflammation and microbial homeostasis. The variant rs2241880 can diminish these capabilities, potentially contributing to inflammatory bowel disease (IBD) pathogenesis.

    OBJECTIVES: To perform an updated meta-analysis on the association between ATG16L1 rs2241880 and IBD susceptibility by exploring the impact of age, ethnicity, and geography. Moreover, to investigate the association between rs2241880 and clinical features.

    METHODS: Literature searches up until September 2022 across 7 electronic public databases were performed for all case-control studies on ATG16L1 rs2241880 and IBD. Pooled odds ratios (ORP ) and 95% CI were calculated under the random effects model.

    RESULTS: Our analyses included a total of 30,606 IBD patients, comprising 21,270 Crohn's disease (CD) and 9336 ulcerative colitis (UC) patients, and 33,329 controls. ATG16L1 rs2241880 was significantly associated with CD susceptibility, where the A allele was protective (ORP : 0.74, 95% CI: 0.72-0.77, p-value: <0.001), while the G allele was a risk factor (ORP : 1.23, 95% CI: 1.09-1.39, p-value: 0.001), depending on the minor allele frequencies observed in this multi-ancestry study sample. rs2241880 was predominantly relevant in Caucasians from North America and Europe, and in Latin American populations. Importantly, CD patients harbouring the G allele were significantly more predisposed to perianal disease (ORP : 1.21, 95% CI: 1.07-1.38, p-value: 0.003).

    CONCLUSIONS: ATG16L1 rs2241880 (G allele) is a consistent risk factor for IBD in Caucasian cohorts and influences clinical outcomes. As its role in non-Caucasian populations remains ambiguous, further studies in under-reported populations are necessary.

    Matched MeSH terms: Carrier Proteins/genetics; Autophagy-Related Proteins/genetics
  2. Fulltext Cell targeting in anti-cancer gene therapy
    Lila MA, Siew JS, Zakaria H, Saad SM, Ni LS, Abdullah JM
    Malays J Med Sci, 2004 Jan;11(1):9-23.
    PMID: 22977356 MyJurnal
    Gene therapy is a promising approach towards cancer treatment. The main aim of the therapy is to destroy cancer cells, usually by apoptotic mechanisms, and preserving others. However, its application has been hindered by many factors including poor cellular uptake, non-specific cell targeting and undesirable interferences with other genes or gene products. A variety of strategies exist to improve cellular uptake efficiency of gene-based therapies. This paper highlights advancements in gene therapy research and its application in relation to anti-cancer treatment.
    Matched MeSH terms: Proteins
  3. Expression of soluble human Neonatal Fc-receptor (FcRn) in Escherichia coli through modification of growth environment
    Ng WK, Lim TS, Lai NS
    Protein Expr Purif, 2016 11;127:73-80.
    PMID: 27412717 DOI: 10.1016/j.pep.2016.07.004
    Neonatal Fc-receptor (FcRn) with its affinity to immunoglobulin G (IgG) has been the subject of many pharmacokinetic studies in the past century. This protein is well known for its unique feature in maintaining the circulating IgG from degradation in blood plasma. FcRn is formed by non-covalent association between the α-chain with the β-2-microglobulin (β2m). Many studies have been conducted to produce FcRn in the laboratory, mainly using mammalian tissue culture as host for recombinant protein expression. In this study, we demonstrate a novel strategy to express the α-chain of FcRn using Escherichia coli as the expression host. The expression vector that carries the cDNA of the α-chain was transformed into expression host, Rosetta-gami 2 strain for inducible expression. The bacterial culture was grown in a modified growth medium which constitutes of terrific broth, sodium chloride (NaCl), glucose and betaine. A brief heat shock at 45 °C was carried out after induction, before the temperature for expression was reduced to 22 °C and grown for 16 h. The soluble form of the α-chain of FcRn expressed was tested in the ELISA and dot blot immunoassay to confirm its native functionality. The results implied that the α-chain of FcRn expressed using this method is functional and retains its pH-dependent affinity to IgG. Our study significantly suggests that the activity of human FcRn remain active and functional in the absence of β2m.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/chemistry
  4. Lipocalin-2 is associated with modulation of disease phenotype in a patient with concurrent JAK2-V617F and BCR-ABL mutation
    Nadarajan VS, Ang CH, Bee PC
    Eur. J. Haematol., 2012 Feb;88(2):175-8.
    PMID: 21950422 DOI: 10.1111/j.1600-0609.2011.01712.x
    We investigated the role of lipocalin-2 (LCN-2) and its receptor (SLC22A17) in mediating clonal dominance in a patient with both BCR-ABL and JAK2-V617F mutations. LCN-2 mRNA showed a near 50-fold increase in expression, accompanied by down-regulation of SLC22A17, coinciding with increase in BCR-ABL transcripts, loss of JAK2-V617F and change of clinical phenotype from polycythaemia vera to chronic myeloid leukaemia. These changes were reversed after commencing imatinib mesylate. Consistent with experimental studies, BCR-ABL+ cells express LCN-2 leading to suppression of BCR-ABL- cells and explain their eventual dominance when occurring together with JAK2-V617F.
    Matched MeSH terms: Acute-Phase Proteins/genetics; Acute-Phase Proteins/metabolism*; Proto-Oncogene Proteins/genetics; Proto-Oncogene Proteins/metabolism*; Fusion Proteins, bcr-abl/genetics*; Organic Cation Transport Proteins/genetics; Organic Cation Transport Proteins/metabolism
  5. Fulltext Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1
    Ruslan R, Abd Rahman RN, Leow TC, Ali MS, Basri M, Salleh AB
    Int J Mol Sci, 2012;13(1):943-60.
    PMID: 22312296 DOI: 10.3390/ijms13010943
    Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
    Matched MeSH terms: Bacterial Proteins/genetics; Bacterial Proteins/metabolism*; Bacterial Proteins/chemistry; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/chemistry
  6. Molecular characterization of tgd057, a novel gene from Toxoplasma gondii
    Wan KL, Chang TL, Ajioka JW
    J. Biochem. Mol. Biol., 2004 Jul 31;37(4):474-9.
    PMID: 15469736
    The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Protozoan Proteins/genetics; Protozoan Proteins/isolation & purification; Protozoan Proteins/metabolism*
  7. Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates
    Tan YP, Ling TC, Yusoff K, Tan WS, Tey BT
    J Microbiol, 2005 Jun;43(3):295-300.
    PMID: 15995649
    In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification*; Recombinant Proteins/metabolism; Viral Proteins/genetics; Viral Proteins/isolation & purification*; Viral Proteins/metabolism
  8. Fulltext Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) from Dermatophagoides farinae
    Pang SL, Ho KL, Waterman J, Teh AH, Chew FT, Ng CL
    Acta Crystallogr F Struct Biol Commun, 2015 Nov;71(Pt 11):1396-400.
    PMID: 26527267 DOI: 10.1107/S2053230X1501818X
    Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/chemistry; Arthropod Proteins/genetics*; Arthropod Proteins/isolation & purification; Arthropod Proteins/chemistry*
  9. Fulltext Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology
    Callaghan PS, Siriwardana A, Hassett MR, Roepe PD
    Malar J, 2016;15(1):186.
    PMID: 27036417 DOI: 10.1186/s12936-016-1238-1
    Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT).
    Matched MeSH terms: Protozoan Proteins; Membrane Transport Proteins
  10. Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents
    Joseph NM, Ho KL, Tey BT, Tan CS, Shafee N, Tan WS
    Biotechnol Prog, 2016 Jul 08;32(4):1038-45.
    PMID: 27088434 DOI: 10.1002/btpr.2279
    The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry; Viral Matrix Proteins/biosynthesis; Viral Matrix Proteins/metabolism; Viral Matrix Proteins/chemistry*
  11. Fulltext Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes
    Abu Bakar F, Yeo CC, Harikrishna JA
    Int J Mol Sci, 2016 Apr 20;17(4).
    PMID: 27104531 DOI: 10.3390/ijms17040321
    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.
    Matched MeSH terms: Bacterial Proteins/genetics*; Bacterial Proteins/metabolism; Recombinant Fusion Proteins/analysis; Recombinant Fusion Proteins/genetics; Green Fluorescent Proteins/analysis
  12. Fulltext Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein
    Rahaman SN, Mat Yusop J, Mohamed-Hussein ZA, Ho KL, Teh AH, Waterman J, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 Mar;72(Pt 3):207-13.
    PMID: 26919524 DOI: 10.1107/S2053230X16002016
    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
    Matched MeSH terms: Proteins/isolation & purification; Proteins/metabolism; Proteins/chemistry*; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/chemistry*
  13. Fulltext Natural Loss of eyeless/Pax6 Expression in Eyes of Bicyclus anynana Adult Butterflies Likely Leads to Exponential Decrease of Eye Fluorescence in Transgenics
    Das Gupta M, Chan SK, Monteiro A
    PLoS One, 2015;10(7):e0132882.
    PMID: 26173066 DOI: 10.1371/journal.pone.0132882
    Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.
    Matched MeSH terms: Eye Proteins/metabolism*; Repressor Proteins/metabolism*; Homeodomain Proteins/metabolism*; Green Fluorescent Proteins/metabolism
  14. Fulltext Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival
    Kong YH, Syed Zanaruddin SN, Lau SH, Ramanathan A, Kallarakkal TG, Vincent-Chong VK, et al.
    PLoS One, 2015;10(7):e0134045.
    PMID: 26214683 DOI: 10.1371/journal.pone.0134045
    Oral squamous cell carcinoma (OSCC) is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer's aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT) is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2) and other genes intimately related to EMT (CDH1 and LAMC2) at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM). Specifically, CDH1 loss was significantly associated with Broder's grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management.
    Matched MeSH terms: Neoplasm Proteins/biosynthesis*; Nuclear Proteins/biosynthesis*; Repressor Proteins/biosynthesis*; Homeodomain Proteins/biosynthesis*
  15. Existence of two emm-like "mrp" and "emm" genes in the mga regulon of the Streptococcus pyogenes strain ST4547
    Eshaghi M, Ali AM, Jamal F, Yusoff K
    J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):23-8.
    PMID: 12186779
    Streptococcus pyogenes ST4547 is an opacity factor negative strain, which has been recently reported as a new emm type from Malaysia. Nucleotide sequencing of the mga regulon of this strain showed the existence of two emm-like genes. The emm gene located upstream of the scpA gene comprises 1305 nucleotides encoding the putative precursor M protein of 435 amino acids in length with an M(r) of 49 kDa. or a predicted mature protein of 394 amino acids with an M(r) of 44.8 kDa. Another gene mrpST4547 was located upstream of the emm gene and downstream of the mga gene. The sequence of this mrp gene comprises 1167 nucleotides encoding a predicted protein of 388 amino acids in length with an M(r) of 42.2 kDa. or a predicted mature protein of 347 amino acids with an M(r) of 37.9 kDa. The mga regulon of strain ST4547 has a mosaic structure comprising segments, which originated from different OF positive and OF negative strains. The sequences flanking the hyper-variable and C repeats of the emmST4547 gene showed high similarity to corresponding regions in the mga regulon of OF positive strains notably M15, M4, M22 and M50. In contrast, the sequence within the hyper-variable and C repeat regions of the emmST4547 gene revealed high similarity to equivalent regions in the OF negative strains. These data indicates that horizontal transfer of emm-like gene could have occurred between OF positive and OF negative strains resulting in architectural divergence in the mga regulon.
    Matched MeSH terms: Bacterial Outer Membrane Proteins/genetics*; Bacterial Proteins*; Carrier Proteins/genetics*; Membrane Proteins/genetics*
  16. Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase
    Kalhori N, Nulit R, Go R
    Protein J, 2013 Oct;32(7):551-9.
    PMID: 24132392 DOI: 10.1007/s10930-013-9516-z
    Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.
    Matched MeSH terms: Plant Proteins/genetics; Plant Proteins/metabolism; Plant Proteins/chemistry*; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry*
  17. Validation of a Burkholderia pseudomallei hypothetical protein and determination of its translational start codon using chromosomal integration of His-Tag coding sequence
    Yam H, Abdul Rahim A, Gim Luan O, Samian R, Abdul Manaf U, Mohamad S, et al.
    Protein J, 2012 Mar;31(3):246-9.
    PMID: 22354666 DOI: 10.1007/s10930-012-9398-5
    In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
    Matched MeSH terms: Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism*; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism
  18. Dengue envelope domain III-peptide binding analysis via tryptophan fluorescence quenching assay
    Baharuddin A, Amir Hassan A, Othman R, Xu Y, Huang M, Ario Tejo B, et al.
    Chem Pharm Bull (Tokyo), 2014;62(10):947-55.
    PMID: 25273053
    In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/chemistry; Viral Envelope Proteins/antagonists & inhibitors*; Viral Envelope Proteins/genetics; Viral Envelope Proteins/metabolism
  19. Application of the linear interaction energy method (LIE) to estimate the binding free energy values of Escherichia coli wild-type and mutant arginine repressor C-terminal domain (ArgRc)-l-arginine and ArgRc-l-citrulline protein-ligand complexes
    Asi AM, Rahman NA, Merican AF
    J Mol Graph Model, 2004 Mar;22(4):249-62.
    PMID: 15177077
    Protein-ligand binding free energy values of wild-type and mutant C-terminal domain of Escherichia coli arginine repressor (ArgRc) protein systems bound to L-arginine or L-citrulline molecules were calculated using the linear interaction energy (LIE) method by molecular dynamics (MD) simulation. The binding behaviour predicted by the dissociation constant (K(d)) calculations from the binding free energy values showed preferences for binding of L-arginine to the wild-type ArgRc but not to the mutant ArgRc(D128N). On the other hand, L-citrulline do not favour binding to wild-type ArgRc but prefer binding to mutant ArgRc(D128N). The dissociation constant for the wild-type ArgRc-L-arginine complex obtained in this study is in agreement with reported experimental results. Our results also support the experimental data for the binding of L-citrulline to the mutant ArgRc(D128N). These showed that LIE method for protein-ligand binding free energy calculation could be applied to the wild-type and the mutant E. coli ArgRc-L-arginine and ArgRc-L-citrulline protein-ligand complexes and possibly to other transcriptional repressor-co-repressor systems as well.
    Matched MeSH terms: Bacterial Proteins*; Repressor Proteins/genetics; Repressor Proteins/metabolism*; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism*
  20. Oncogenic effects of WNT5A in Epstein-Barr virus‑associated nasopharyngeal carcinoma
    Yap LF, Ahmad M, Zabidi MM, Chu TL, Chai SJ, Lee HM, et al.
    Int J Oncol, 2014 May;44(5):1774-80.
    PMID: 24626628 DOI: 10.3892/ijo.2014.2342
    The molecular events that drive the progression of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) are still to be elucidated. Here, we report for the first time the pathogenic significance of an NPC-associated gene, wingless-type MMTV integration site family, member 5A (WNT5A) and the contribution of EBV to its expression. WNT5A is a representative Wnt protein that activates non-canonical Wnt signalling. With regard to its role in carcinogenesis, there is conflicting evidence as to whether WNT5A has a tumour-promoting or tumour-suppressive role. We show that WNT5A is upregulated in primary NPC tissue samples. We also demonstrate that WNT5A expression was dramatically increased in NPC cell lines expressing the EBV-encoded LMP2A gene, suggesting that this EBV-encoded latent gene is responsible for upregulating WNT5A in NPC. In addition, in vitro WNT5A overexpression promotes the proliferation, migration and invasion of NPC cells. Our results not only reveal pro-tumorigenic effects of WNT5A in NPC but also suggest that WNT5A could be an important therapeutic target in patients with EBV-associated disease.
    Matched MeSH terms: Proto-Oncogene Proteins/genetics; Proto-Oncogene Proteins/metabolism*; Viral Matrix Proteins/metabolism*; Wnt Proteins/genetics; Wnt Proteins/metabolism*
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