Biochemical characterization of human and yeast choline and ethanolamine kinases [QD415. S451 2006 f rb].

Kolina kinase (CK) dan etanolamina kinase (EK) merupakan enzim pertama di dalam biosintesis fosfatidilkolina dan fosfatidiletanolamina. Peningkatan aktiviti CK telah dikaitkan dengan tumor manusia. Oleh itu, perencatan CK telah dicadangkan sebagai strategi antikanser yang berpotensi. Choline kinase (CK) and ethanolamine kinase (EK) are the first enzymes in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine. Increased activity of CK has been implicated in human tumors. CK inhibition has therefore been proposed as a potential anticancer strategy

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

AidP, a novel N-Acyl Homoserine Lactonase gene from Antarctic Planococcus sp.

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions

Biochemical Characterization Of Human And Yeast Choline And Ethanolamine Kinases

Kolina kinase (CK) dan etanolamina kinase (EK) merupakan enzim pertama di dalam biosintesis fosfatidilkolina dan fosfatidiletanolamina. Choline kinase (CK) and ethanolamine kinase (EK) are the first enzymes in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine

Effects of protein kinase a phosphorylation on the biochemical properties and subcellular location of human choline kinase beta

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, cka and ckp, which produce three isoforms, CKa1, CKa2, and CKp. Previous studies have associated ckp with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckp has never been elucidated. In this report, the distal promoter region of the ckp 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 ckp promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckp promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckp promoter activity through Ets and GATA elements. PMA also decreased the ckp 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 PKCe or PKCq as the PKC isozyme involved in the PMA-mediated repression of ckp promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCe as the isozyme that mediated the PMA repression of ckp promoter

Pengaruh Tingkah Laku Pengguna Terhadap Kualiti Produk Dan Industri Akuakultur Di Pulau Pinang, Malaysia

Industri akuakultur adalah antara industri terbesar untuk membekalkan bekalan makanan laut di dunia ini. Permintaan terhadap makanan laut yang semakin meningkat menyebabkan berlaku ekploitasi sumber laut secara rakus. Namun akuakultur merupakan salah satu cara untuk mengimbangi permintaan dan penawaran makanan laut di pasaran serta mengurangkan tekanan terhadap penerokaan sumber laut. Walau bagaimanapun, aktiviti akuakultur tanpa kawalan ternyata memberi kesan kepada kualiti persekitaran, dan kualiti produk. Kesedaran yang tinggi dan adanya tekanan dari pihak pengguna dapat meningkatkan daya saing dalam industri akuakultur serta kelestarian dalam system pengeluaran akuakultur. Oleh itu, kajian memilih kawasan kajian di Pulau Pinang untuk merungkai persoalan yang berkaitan dengan pengaruh pengguna terhadap kualiti produk dan industri akuakultur

Cloning, characterization and activity analysis of human choline kinase promoters

Choline kinase (CK) is the first enzyme in the COP-choline pathway, a de novo biosynthetic pathway for major phospholipid in the membrane of eukaryotic cells i.e phosphatidylcholine (Lykidis et a/., 2001 ). This enzyme catalyzes the phosphorylation of choline by ATP to form phosphocholine. In mammalian cells, choline kinase exists as three isoforms that are encoded by two separate genes named cka and ck/3. ck/3 codes for a single protein (CKJ3) while cka undergoes alternative splicing to produce CKu1 and CKu2 isoforms (Malito eta/., 2006). Increased activity of CK and its product, phosphocholine, have been implicated in human carcinogenesis. Elevated phosphocholine level is a common feature in cell lines derived from human tumors and this parameter seems to be able to distinguish malignant cell lines from normal cell lines irrespective of their proliferation rates (Bhakoo eta/., 1996; Aboagye and Bhujwalla, 1999). Overexpression of CK has been reported in a variety of human cancers such as lung, colo rectal as well as prostate adenocarcinomas (Nakagami et a/., 1999, Ramirez de Molina eta/., 2002a, Ramirez de Molina eta/., 2002b). In addition, studies had demonstrated the increased of CK activity upon induction of the H-ras oncogene in mouse fibroblast cell lines. Inhibition of CK has been proposed to be a potential antitumor strategy (Rodriguez-Gonzalez eta/., 2004). Rodriguez-Gonzalez et al. (2004) demonstrated that CK inhibitors could become potent antitumor drugs both in vitro and in vivo. Recently, CKu protein levels have been found to be drastically increased in both human tumors and cell lines derived from human tumor, when compared to normal tissues or appropriate human primary cells, respectively (Aoyama et a/., 2004). Increased levels of CKu mRNA but not CKJ3 in tumor-derived cell lines was also reported (Gallego-Ortega eta/., 2009)

Planococcus versutus sp. nov., isolated from soil

A taxonomic study was performed on a novel Gram-stain-positive, coccus-shaped, orange-pigmented motile bacterium, designated as strain L10.15T. The organism was isolated from a soil sample collected in Lagoon Island (close to Adelaide Island, western Antarctic Peninsula) using a quorum-quenching enrichment medium. Growth occurred at 4–30 °C, pH 6–11 and at moderately high salinity (0–15 %, w/v, NaCl), with optimal growth at 26 °C, at pH 7–8 and with 6 % (w/v) NaCl. 16S rRNA gene sequence analysis showed that strain L10.15T belonged to the genus Planococcus and was closely related to Planococcus halocryophilus Or1T (99.3 % similarity), Planococcus donghaensis JH1T (99.0 %), Planococcus antarcticus DSM 14505T (98.3 %), Planococcus plakortidis AS/ASP6 (II)T (97.6 %), Planococcus maritimus TF-9T (97.5 %), Planococcus salinarum ISL-6T (97.5 %) and Planococcus kocurii NCIMB 629T (97.5 %). However, the average nucleotide identity-MUMmer analysis showed low genomic relatedness values of 71.1–81.7 % to the type strains of these closely related species of the genus Planococcus . The principal fatty acids were anteiso-C15 : 0, C16 : 1ω7c and anteiso-C17 :  0, and the major menaquinones of strain L10.15T were MK-5 (48 %), MK-6 (6 %) and MK-7 (44 %). Polar lipid analysis revealed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and aminophospholipid. The DNA G+C content was 39.4 mol%. The phenotypic and genotypic data indicate that strain L10.15T represents a novel species of the genus Planococcus , for which the name Planococcus versutus sp. nov. is proposed. The type strain is L10.15T (=DSM 101994T=KACC 18918T)

The Influence of Parent-Child Attachment and Peer Attachment on Adolescent Aggressive Behavior

This study aims to determine the role of parent-child attachment and peer attachment on adolescent aggressive behavior. This research is quantitative research. The study population was private high school students in East Mesuji. Using the Krejcie and Morgan formula, a sample of 113 students was selected. Data were collected with three scales, namely the parent-child attachment scale, peer attachment, and aggressive behavior. Subject responses were analyzed using SPSS version 25 and analyzed with multiple linear regression models, stimulant testing (F test) resulted in a significance value of 0.000 (<0.05). Thus, the results of this study indicate that parent-child attachment and peer attachment together have a very significant influence on adolescent aggressive behavior.

Highly Specific Antibodies for Co-Detection of Human Choline Kinase α1 and α2 Isoforms

BACKGROUND: Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples. CONCLUSION/SIGNIFICANCE: Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels