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  1. Cloning of triose phosphate isomerase gene from an antarctic psychrophilic Pseudomonas sp. by degenerate and splinkerette PCR
    See Too WC, Few LL
    World J Microbiol Biotechnol, 2010 Jul;26(7):1251-9.
    PMID: 24026930 DOI: 10.1007/s11274-009-0295-9
    Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.
  2. Fulltext ChoK-ing the Pathogenic Bacteria: Potential of Human Choline Kinase Inhibitors as Antimicrobial Agents
    Khalifa M, Few LL, See Too WC
    Biomed Res Int, 2020;2020:1823485.
    PMID: 32695809 DOI: 10.1155/2020/1823485
    Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S. pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs.
  3. Fulltext Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway
    Kuan CS, Yee YH, See Too WC, Few LL
    PLoS One, 2014;9(12):e113485.
    PMID: 25490397 DOI: 10.1371/journal.pone.0113485
    Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.
  4. Fulltext UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines
    Chua SL, See Too WC, Khoo BY, Few LL
    Cytotechnology, 2011 Dec;63(6):645-54.
    PMID: 21850463 DOI: 10.1007/s10616-011-9383-4
    Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C(t) values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.
  5. Fulltext Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition
    Chang CC, Few LL, Konrad M, See Too WC
    PLoS One, 2016;11(5):e0154702.
    PMID: 27149373 DOI: 10.1371/journal.pone.0154702
    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.
  6. Fulltext Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells
    Kuan CS, See Too WC, Few LL
    PLoS One, 2016;11(1):e0147886.
    PMID: 26807725 DOI: 10.1371/journal.pone.0147886
    Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.
  7. Downregulation of human choline kinase α gene expression by miR-876-5p
    Ayub Khan SM, Few LL, See Too WC
    Mol Med Rep, 2018 May;17(5):7442-7450.
    PMID: 29568919 DOI: 10.3892/mmr.2018.8762
    Choline kinase (CK) is the first enzyme in the CDP-choline pathway for the synthesis of phosphatidylcholine, the most abundant phospholipid in the mammalian cell membrane. This enzyme exists as three isozymes (α1, α2 and β) and the CKα isozyme has been implicated in cancer pathogenesis. Inhibition of CK activity has been proposed for cancer therapies. MicroRNAs (miRNAs/miRs) are non‑coding RNAs that serve important roles in diverse biological pathways and human diseases, including cancer. However, the regulation of CKα gene expression by miRNAs has never been investigated, to the best of the authors' knowledge. In the present study, two miRNA mimics, miR‑876‑5p and miR‑646, were transfected into the HepG2 cell line and the effect of these miRNAs on the levels of CKα mRNA were determined by reverse transcription‑quantitative polymerase chain reaction. Cells transfected with 25 nM miR‑876‑5p for 48 h exhibited significantly lower levels of CKα mRNA. Following optimization, miR‑876‑5p caused four times lower levels of CKα mRNA compared to the negative control. Effects of the miRNAs on HepG2 cell viability and cellular morphology were additionally analyzed using an MTT cell viability assay and scanning electron microscopy, respectively. HepG2 cells that were transfected with the optimum concentration of miR‑876‑5p for the optimum duration exhibited 25% lower viability than negative control and signs of apoptosis in electron micrographs. The results suggested miR‑876‑5p as a potential miRNA modulator of CKα expression in the cells, and may be relevant for the design of more effective anticancer strategy targeting CK.
  8. Inhibition of choline kinase as an antiamoebic approach in Entamoeba histolytica infection
    Teh ZH, Lim BH, See Too WC, Few LL
    Trop Biomed, 2023 Dec 01;40(4):430-438.
    PMID: 38308830 DOI: 10.47665/tb.40.4.008
    Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.
  9. Identification and Characterization of Entamoeba histolytica Choline Kinase
    Chang CH, See Too WC, Lim BH, Few LL
    Acta Parasitol, 2024 Mar;69(1):426-438.
    PMID: 38172465 DOI: 10.1007/s11686-023-00763-1
    PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future.

    METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes.

    RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting.

    CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

  10. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples
    Foo PC, Chan YY, See Too WC, Tan ZN, Wong WK, Lalitha P, et al.
    J Med Microbiol, 2012 Sep;61(Pt 9):1219-1225.
    PMID: 22556327 DOI: 10.1099/jmm.0.044552-0
    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.
  11. Fulltext DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells
    Mohamed Sa'dom SAF, Raikundalia S, Shamsuddin S, See Too WC, Few LL
    Genes (Basel), 2021 06 01;12(6).
    PMID: 34205960 DOI: 10.3390/genes12060853
    Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between -225 and -56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.
  12. Unusual metal ion cofactor requirement of Entamoeba histolytica choline and ethanolamine kinase isoforms
    Chang CH, Few LL, Lim BH, Yvonne-Tee GB, Chew AL, See Too WC
    Parasitol Res, 2023 Jul;122(7):1651-1661.
    PMID: 37202563 DOI: 10.1007/s00436-023-07869-5
    The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn2+ over the typical Mg2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn2+, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg2+. Specifically, in Mg2+, EhCK1 exhibited a Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km for choline and ATP were higher than in equimolar of Mg2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn2+, exhibiting Michaelis-Menten kinetic with ethanolamine (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg2+, while choline kinase β differentially recognized choline and ethanolamine in Mg2+ and Mn2+, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.
  13. Fulltext Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease
    Fong SW, Few LL, See Too WC, Khoo BY, Nik Ibrahim NN, Yahaya SA, et al.
    BMC Res Notes, 2015;8:679.
    PMID: 26576922 DOI: 10.1186/s13104-015-1677-8
    Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated.
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