Kozo Ishidate

Expression and characterization of the active molecular forms of choline/ethanolamine kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver

Choline}ethanolamine kinase (ChoK}EtnK) exists as at least three isoforms (α1, α2 and β) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northernand Western-blot analyses indicated that either the α (α1 plus α2) or the β isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the α isoform was highest in testis, whereas that for the β isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of α}β hetero-oligomers, with relatively small parts of activity expressed by α}α and β}β homo-oligomers. This is the first

EFFECTS OF FUSOGENIC AND DNA-BINDING AMPHIPHILIC COMPOUNDS ON THE RECEPTOR-MEDIATED GENE TRANSFER INTO HEPATIC CELLS BY ASIALOFETUIN-LABELED LIPOSOMES

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta-Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF-liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF-liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta-Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2-fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner.

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

Abstract The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.

Choline/ethanolamine kinase from rat kidney.

Publisher Summary Choline kinase (ATP:choline phosphotransferase) and ethanolamine kinase (ATP:ethanolamine O-phosphotransferase) catalyze the phosphorylation of choline/ethanolamine by ATP in the presence of Mg 2+ , yielding phosphocholine/phosphoethanolamine and ADP. This enzyme step commits choline (ethanolamine) to the CDPcholine (CDPethanolamine) pathway for the biosynthesis of phosphatidylcholine (phosphatidylethanolamine) in all animal cells. The chapter discusses different methods to assay choline kinase activity. The purification of choline/ethanolamine kinase from rat kidney is also discussed. Purification is started with the high-speed supernatant fraction from fresh kidneys (∼200 g wet weight). All procedures are carried out below 5 ° C. One of the most important features of choline/ethanolamine kinase is its inducibility in various experimental systems. Although the physiological significance of the inducibility has not yet been fully understood, the induction of choline/ethanolamine kinase by several means suggests that more than one mechanism could be involved in this process. Distinct mechanisms of choline kinase induction have been demonstrated between rooster liver by estrogens and rat liver by certain hepatotoxins. A new isozyme of choline/ethanolamine kinase appears to be preferentially induced in the latter case.

MOLECULAR CLONING OF MOUSE CHOLINE KINASE AND CHOLINE/ETHANOLAMINE KINASE : THEIR SEQUENCE COMPARISON TO THE RESPECTIVE RAT HOMOLOGS

Complementary DNAs homologous to a rat 42-kDa choline/ethanolamine kinase [C. Aoyama et al., Biochim. Biophys. Acta 1390 (1998) 1-7] and to a 50-kDa choline kinase [T. Uchida and S. Yamashita, J. Biol. Chem. 267 (1992) 10156-10162] were isolated from a 17-day post coitum mouse embryo cDNA library and their sequences were compared with the two murine species, respectively. The nucleotide sequence homology (within the coding sequence) between mouse and rat 50-kDa choline kinases (96.0%) was considerably higher than that between their 42-kDa choline/ethanolamine kinases (92.4%). Northern blot and RT-PCR studies on several rat tissues demonstrated that both isozymes are expressed ubiquitously with the highest level in testis.

Complementary DNA sequence for a 42 kDa rat kidney choline/ethanolamine kinase.

By means of peptide sequence information, several cDNA clones encoding a 42 kDa choline/ethanolamine kinase were isolated from a rat kidney cDNA library. Eight clones were sequenced with all of them resulting in identical overlapping nucleotide sequences. Four of them possessed entire open reading frame which could encode 394 amino acids with a calculated molecular size of 45 100. The predicted amino acid sequence contained all of the internal peptide fragment sequences derived from the purified 42 kDa enzyme. When the open reading frame was introduced into pGEX-2T vector and transfected into E. coli cells, a significant choline/ethanolamine kinase activity did appear in the cell lysate. A homology comparison with the previously reported choline kinase cDNAs (CKR1 and CKR2) from rat liver showed 66%-68% in entire nucleotide sequences and 57%-59% in amino acid sequences, indicating that the cloned cDNA here must be a novel CK/EK gene product. (c) 1998 Elsevier Science B. V.

Actinomycin D-sensitive induction of choline kinase by carbon tetrachloride intoxication in rat liver

A single intraperitoneal dose(1 ml/kg body weight) of carbon tetrachloride (CCl4) caused a rapid and drastic induction of choline kinase activity in rat liver cytosol. The administration of either cycloheximide or actinomycin D completely blocked the CCl4-mediated induction of choline kinase activity, indicating that the elevated activity could be due to the change in the enzyme level. The pretreatment of rats with phenobarbital did not cause any significant effect on hepatic choline kinase induction by CCl4, suggesting that the induction may not be directly related to the metabolic rate of CCl4. A considerable part of induced form(s) of choline kinase appeared not to be a form present in the liver of untreated rats. The contribution of adrenals to the CCl4-mediated hepatic choline kinase induction could be ruled out.

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

Abstract The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [ 32 P]orthophosphate, [Me− 14 C]choline or [2− 3 H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [ 32 P] and [ 14 C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [ 32 P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2− 3 -H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [ 32 P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver: II. Its relation to net phosphatidylcholine biosynthesis

The effect of a single dose (50 mg/kg body weight) of 3-methylcholanthrene on de novo phosphatidylcholine biosynthetic activities in rat liver was studied both in a cell-free system and with slice experiments. 3-Methylcholanthrene caused a significant depression of either [methyl-14C]choline or [2-(3)H]glycerol incorporation into phosphatidylcholine when the precursor was incubated with liver slices. At the same time, there occurred a significant accumulation of radioactivity in either cholinephosphate or diacylglycerol molecule from [14C]choline or [3H]glycerol, respectively, suggesting that 3-methylcholanthrene could cause an inhibitory effect on hepatic phosphatidylcholine synthesis at the cholinephosphotransferase or/and cholinephosphate cytidylyltransferase step. Subsequent studies, where the activities of the three enzymes involved in de novo phosphatidylcholine synthesis were compared between control and 3-methylcholanthrene-pretreated rat liver subcellular fractions, demonstrated that the cholinephosphotransferase step could be the site of inhibition by 3-methylcholanthrene. On the other hand, 3-methylcholanthrene caused a significant induction of choline kinase activity in a time-dependent manner and, at the same time, the cholinephosphate pool size in liver cytosol was enlarged 2-3-fold when compared to the respective control. The overall results suggested strongly that 3-methylcholanthrene causes the counteractive effects on the de novo phosphatidylcholine biosynthesis, induction of choline kinase activity and inhibition of cholinephosphotransferase activity, both of which could participate in a concomitant increase in cholinephosphate pool size in rat liver.

Sialadenitis in patients with chronic hepatitis C is not directly related to hepatitis C virus

Sjogrens syndrome has been suspected to be an extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. To evaluate the association of sialadenitis with HCV infection, serum levels of salivary amylase (s-isoamylase) and antibodies to Ro (SS-A) and La (SS-B) were analyzed in 114 patients with chronic hepatitis C. Serum s-isoamylase levels were monitored before and after HCV was eradicated by interferon therapy. Immunohistochemistry and Western blotting using anti-HCV antibodies, and in situ hybridization of HCV-RNA were performed in the salivary gland. Serum s-isoamylase levels were elevated in patients with chronic hepatitis C (P<0.0001). The s-isoamylase remained high even after HCV was eradicated. The in situ hybridization did not show the presence of HCV-RNA in the salivary gland from patients with chronic hepatitis C. A protein reacting with anti-HCV-E2 antibodies was found in the cytosol fraction of normal salivary gland from HCV-negative cases. Latent sialadenitis is frequently observed in chronic hepatitis C, which is not directly related to HCV per se. The presence of a common epitope between antigenic protein in the salivary gland and the HCV-derived protein may be a possible pathogenetic mechanisms such as molecular mimicry.