AP4 transcription factor binding site is a repressor element in ek2 promoter of human liver carcinoma cell line, HepG2

Ethanolamine kinase (EK) is the first enzyme in the Kennedy pathway for the biosynthesis of phosphatidylethanolamine. Although EK has been reported to be involved in phospholipid biosynthesis, carcinogenesis, cell growth, muscle development and sex determination during embryonic development, little is known about its transcriptional regulation by endogenous or exogenous signals. Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. Compared to ek1 gene, ek2 is expressed at a higher level in liver and EK2 isoforms also accept choline as substrate besides ethanolamine, which could contribute to liver carcinogenesis. The main aim of this study was to analyze and characterize the human ek2 promoter in cultured mammalian cells. Human ek2 (2011 bp) promoter was cloned into reporter vector, pGL4.10 [luc2] and the promoter activities were studied in human liver carcinoma (HepG2 cells). Sequence analyses showed that ek2 promoter contains numerous putative transcription factor binding sites including AP4 and it is devoid of a recognizable consensus TATA box but it contains a high number of guanine (G) and cytosine (C) nucleotides. PCR mutagenesis of three nucleotides at E-box motif of AP4 transcription binding site located between -293 and -276 of ek2 promoter was successfully performed to show that AP4 transcription factor binding site acts as a repressive element in the regulation of ek2 expression. AP4 upregulation has been implicated in bad prognosis of carcinoma, therefore the regulatory role of AP4 binding site reported in this study could be a link between ek2 and carcinogenesis. Although further studies need to be carried out to understand and to determine the repression mechanism of AP4 in ek2 promoter, the characterization and analysis of ek promoter performed in this study provide important understanding of its basal transcriptional regulation which would allow us to control ek expression levels in pathologic conditions that involve this gene

Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578

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

Antimicrobial resistance of Listeria monocytogenes and Salmonella Enteritidis isolated from vegetable farms and retail markets in Malaysia

Listeriosis and salmonellosis are the major foodborne illnesses worldwide. Over the last decade, increasing reports about the antibiotic resistance of Listeria monocytogenes and Salmonella from diverse sources have prompted public health concerns, especially in developing countries with over reliance or misuse of antibiotic drugs in the treatment of humans and animals. In this study, antibiotic susceptibility profiles of 58 L. monocytogenes and 12 Salmonella Enteritidis strains from vegetable farms and retail markets in Malaysia were tested by the standard disk diffusion method. Listeria monocytogenes isolates were found to exhibit 100% resistance to penicillin G. Also, high resistance patterns were observed for meropenem (70.7%) and rifampicin (41.4%). The multiple antibiotic resistance (MAR) index of L. monocytogenes isolates ranged from 0.11 to 0.56. Besides, the antibiogram results revealed that multidrug-resistant (MDR) S. Enteritidis were detected and all the S. Enteritidis isolates demonstrated resistance to at least four antibiotics. Ampicillin, amoxicillin, and trimethoprim failed to inhibit all the S. Enteritidis strains. Salmonella Enteritidis isolates also displayed high resistance to nalidixic acid (75.0%), trimethoprim-sulfamethoxazole (75.0%), and chloramphenicol (66.7%). Findings in this study indicated that vegetables could be potential sources of multidrug resistance of L. monocytogenes and S. Enteritidis, which can be a serious issue and a major concern for public health. Thus, there is a great need for surveillance programs in Malaysia to continuously monitor the antibiotic resistance profiles of important pathogens

Simulation of decontamination and transmission of Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes during handling of raw vegetables in domestic kitchens

Epidemiological data indicates that a large number of foodborne illnesses are attributed to cross-contamination during food preparation in the domestic kitchen. The objectives of this study were to evaluate the efficiency of household washing practices in removing Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Enteritidis on artificially contaminated lettuce and to determine the transfer rate of these three foodborne pathogens from contaminated lettuce to wash water, tomato, cabbage, and cutting boards during washing and cutting processes. Washing under the running tap water with scrubbing for 60 s was the most effective method in reducing pathogen populations by 1.86 to 2.60 log10 CFU/g. Also, final rinsing and scrubbing practices were found to enhance the efficiency of washing treatment. In this study, the transfer rates of S. Enteritidis, E. coli O157:H7, and L. monocytogenes from cutting board to cabbage and tomato via cutting process (17.5 to 31.7%) were higher (P<0.05) than from wash water to cabbage and tomato (0.8 to 23.0%) during washing treatment. Overall, our findings suggest that wash water and cutting board can be potential vehicles in the dissemination of foodborne pathogens. Therefore, there is a need to promote consumer awareness for proper handling practices in the kitchen to minimise the risk of foodborne infection

Simulation of decontamination and transmission of Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes during handling of raw vegetables in domestic kitchens

Epidemiological data indicates that a large number of foodborne illnesses are attributed to cross-contamination during food preparation in the domestic kitchen. The objectives of this study were to evaluate the efficiency of household washing practices in removing Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Enteritidis on artificially contaminated lettuce and to determine the transfer rate of these three foodborne pathogens from contaminated lettuce to wash water, tomato, cabbage, and cutting boards during washing and cutting processes. Washing under the running tap water with scrubbing for 60 s was the most effective method in reducing pathogen populations by 1.86–2.60 log10 CFU/g. Also, final rinsing and scrubbing practices were found to enhance the efficiency of washing treatment. In this study, the transfer rates of S. Enteritidis, E. coli O157:H7, and L. monocytogenes from cutting board to cabbage and tomato via cutting process (17.5–31.7%) were higher (P < 0.05) than from wash water to cabbage and tomato (0.8–23.0%) during washing treatment. Overall, our findings suggest that wash water and cutting board can be potential vehicles in the dissemination of foodborne pathogens. Therefore, there is a need to promote consumer awareness for proper handling practices in the kitchen to minimise the risk of foodborne infection

Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells.

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.In this report, we mapped the important regulatory regions in the human ek1 promoter. 5' deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells.In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner

Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway.

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.In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors

Effect of TSA on the activities of wild type and Sp(-40/-31) mutated <i>ek1</i> minimal promoters.

<p>A. Promoter activity of pGL4.10-<i>ek1</i>(-69/+1) reporter construct in HCT116 cells treated with the indicated concentrations of TSA for 24 hours (*<i>p</i> < 0.001; **<i>p</i> < 0.01; ***<i>p</i> < 0.05 vs. DMSO control; #<i>p</i> < 0.05, significant within TSA treatment group). B. Promoter activity of pGL4.10-<i>ek1</i>(-69/+1) reporter construct in HCT116 cells treated with 1 μM of TSA for the indicated time points (*<i>p</i> < 0.05 vs. DMSO control). C. Activities of wild type <i>ek1</i> minimal promoter and Sp(-40/-31)-mutated <i>ek1</i> minimal promoter after treatment with 1 μM of TSA for 24 hours. Each bar represents the mean ± SEM of triplicate samples from three independent experiments. D. Effect of TSA on <i>ek1</i> gene expression in HCT116 cells. HCT116 cells were treated with 1 μM of TSA for 24 hours (**<i>p</i> < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.</p

Distributed under Creative Commons CC-BY 4.0 Genomic insight into pathogenicity of dematiaceous fungus Corynespora cassiicola

ABSTRACT Corynespora cassiicola is a common plant pathogen that causes leaf spot disease in a broad range of crop, and it heavily affect rubber trees in Malaysia (Hsueh, 2011

Primers used for generating promoter-luciferase constructs and PCR site-directed mutagenesis.

<p>Primers used for generating promoter-luciferase constructs and PCR site-directed mutagenesis.</p