Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (alpha1, alpha2 and beta) that are encoded by two separate genes termed ck-alpha and ck-beta. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-alpha1 and CK-beta tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424-K430 for CK-alpha1 and Q379-K385 for CK-beta) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-alpha1 was found to be essential for alpha1/alpha1 homo-dimerization but not for alpha1/beta hetero-dimerization. A mutation of the corresponding D276 of CK-beta to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.

In the present study, it was showed that combination with the plasmid DNA of genes under the control of α-fetoprotein (AFP) promoter and asialofetuin-labelled cationic liposomes (AF-liposomes) achieved specific and enhanced gene expression in AFP high-producing HCC cells. The plasmids, containing luciferase gene (pAFP-luc) or herpes simplex virus thymidine kinase gene (pAFP-tk) under the human AFP enhancer/silencer/promoter sequence, were complexed with AF-liposomes and transfected to established cancer cell lines. The transfection of pAFP-luc with AF-liposomes led to higher luciferase expression than that with control cationic liposomes in AFP high-producing hepatocellular carcinomas (HCC). Conversely, almost no expression was observed in AFP non-producing cell lines. Exposure of HepG2 cells to acyclovir following transfection of pAFP-tk with AF-liposomes decreased viability of the cells significantly. These results suggest AF-liposomes significantly enhance specific transgene expression driven by AFP promoter and have the potentiality of a non-viral vector in gene therapy for AFP-producing HCC.

Choline kinase (CK) catalyzes the first phosphorylation reaction in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC), yielding phosphocholine (P-Cho) from choline and ATP in the presence of Mg2+. This enzyme exists in mammalian cells as at least three isoforms that are encoded by two separate genes termed ck-α and ck-β. Each isoform is not active in its monomeric form. The active enzyme consists of either their homo- or hetero-dimeric (or oligomeric) forms. In recent years, the roles of CK in cell growth and cell stress/defense mechanisms have been intensely investigated. These functions of CK do not seem to be directly related to the net PC biosynthesis but predict another important role of this enzyme in certain cell physiology. This review summarizes briefly the recent progress of mammalian CK study which will include the gene structure of each isoform and its possible transcriptional regulation, the active configuration of the enzyme, induction of the particular isoform in chemically induced cell stress, and the possible role of this enzyme as well as of its reaction product, P-Cho, in cell growth and other cellular physiology.

Sjogren's 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.

Cytotoxic T cells (CTLs) are believed to play an important role in the pathogenesis of chronic hepatitis C based on histological findings of the liver and in vivo experiments. Fas-ligand-Fas and perforin dependent pathways are two major killing systems when CTLs induce their target-cell death. Thus, the present study attempts to determine whether these pathways are activated, and if they are, how they are related in chronic hepatitis C. To investigate the expression of Fas-ligand and perforin, we performed double immunofluorescent staining of liver biopsy specimens from patients with chronic hepatitis C. Fas-ligand and perforin expression was observed in mononuclear cells, and the partial coexistence of the two proteins was observed. Cells expressing both proteins were positive for CD45RO(+) T cells (active T cells), whereas cells expressing perforin were negative for CD68 (macrophages). In the cases which had sustained negative HCV-RNA over 6 months after interferon treatment, Fas-ligand was not expressed, although perforin was slightly detectable. To quantitatively assess the balance of these pathways, hepatic mRNAs of Fas-ligand and perforin were measured by quantitative RT-PCR. The ratio of Fas-ligand-mRNA/perforin-mRNA was significantly correlated with serum alanine aminotransferase (ALT) levels (r=0.913). These results suggest that both pathways are activated in chronic hepatitis C and that Fas-ligand-Fas pathway may be predominant in active inflammation.

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (alpha1, alpha2 and beta) 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 Northern- and Western-blot analyses indicated that either the alpha (alpha1 plus alpha2) or the beta isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the alpha isoform was highest in testis, whereas that for the beta 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 alpha/beta hetero-oligomers, with relatively small parts of activity expressed by alpha/alpha and beta/beta homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl(4)) was shown to induce ChoK activity 2-4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl(4) was alpha, not beta. The level of alpha mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the alpha isoform. Consequently, the composition of alpha/alpha homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl(4)-induced liver, whereas it was less than 20% in normal uninduced liver.

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.

Choline/ethanolamine kinase (CK/EK) is the first enzyme in phosphatidylcholine/phosphatidylethanolamine biosynthesis in all animal cells. The highly purified CKs from mammalian sources and their recombinant gene products so far were all shown to have EK activity also, indicating that both activities reside on the same protein. CK/EK in most animal cells exists as several isoforms, for two of which (alpha and beta) their cDNAs have been cloned from both the rat and mouse, and they are found to be separate gene products. The physiological significance for the existence of more than one CK/EK enzyme, however, remains to be clarified. In this study, we isolated mouse genes encoding both types of CK/EK isozyme and determined their entire structure. The 5'-flanking promoter regions were found to have quite different features from each other, indicating that their expression could be under distinct control. Comparison of the nucleotide sequence between the corresponding coding exons showed the best homology (75%) residing on exon VIII. A search of the database resulted in the possible existence of 17 different origins of eukaryotic CK and/or EK, each of which presumably contained the entire amino acid sequence. Multialignment of their putative amino acid sequences led to an identification of the novel consensus sequence possibly required for the expression of either CK or EK activity, which corresponded to the sequence within exons VII and VIII of CK/EK-alpha and -beta genes from the mouse. This sequence was localized in close proximity to the C-terminal region of the general (Brenner's) phosphotransferase concensus sequence which was also completely conserved in all of the putative eukaryotic CK/EK proteins. The results demonstrated that, while both CK/EK-alpha and -beta genes were composed of 11 major exons, the size of their genes was quite different: 40 kb for CK/EK-alpha, whereas it was only 3.5 kb for CK/EK-beta.

BACKGROUND/AIMS Tumor necrosis factor-alpha (TNF-alpha) is believed to play a role in hepatic allograft rejection. However, the specific cellular population responsible for TNF-alpha production during hepatic allograft rejection is not known. Circulating monocyte-macrophage cells are the primary systemic sources of TNF-alpha. In the liver, Kupffer cells are the main producers of TNF-alpha. In this study, we determined which cells are involved in TNF-alpha production during allograft rejection after orthotopic liver transplantation. METHODS In situ hybridization was used to identify cells with TNF-alpha mRNA in the liver. Immunohistochemical staining with ED2 and ED3 was used to differentiate between cellular types (Kupffer cells versus infiltrating monocytes). To detect DNA fragmentation in liver cells, TdT-mediated biotin-dUTP nick-end labeling (TUNEL) was done. Studies were performed in the rat liver transplant model using rejecting (ACI to LEW) and non-rejecting (ACI to ACI) donor/recipient combinations. RESULTS In the control group, cells with TNF-alpha mRNA were rarely observed. In the rejection group, TNF-alpha mRNA was observed in mononuclear cells that were mainly within the vessels of the portal region and occasionally in the sinusoids. The cells with the signals for TNF-alpha mRNA were ED2-negative and ED3-positive. DNA fragmentation was observed in hepatocytes as well as infiltrating mononuclear cells. CONCLUSIONS The main producer of TNF-alpha may be infiltrating mononuclear cells such as monocyte-macrophage cells rather than Kupffer cells during allograft rejection after liver transplantation. Circulating monocyte-macrophages may play a role in the control of allograft rejection.

The chain length distribution of murein glycan strands was analyzed in wild-type cells and in cells in which preseptal and/or septal murein synthesis was prevented in ftsZ84 and ftsI36 mutants of E. coli. This revealed a significant change in glycan chain lengths in newly synthesized murein associated with inactivation of the ftsZ gene product but not with inactivation of the ftsI gene product. This is the first reported abnormality in murein biosynthesis associated with mutation of an essential cell division gene.

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.

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.

The binding of hemimethylated oriC to Escherichia coli membranes has been implicated in the prevention of premature reinitiation at newly replicated chromosomal origins in a reaction that involves the SeqA protein. We describe the resolution of the membrane-associated oriC -binding activity into two fractions, both of which are required for the high-affinity binding of hemimethylated oriC . The active component in one fraction is identified as SeqA. The active component of the second fraction is a previously undescribed protein factor, SeqB. The reconstituted system reproduced the salient characteristics of the membrane-associated binding activity, suggesting that the membrane-associated oriC -binding machinery of E. coli is likely to be a multiprotein system that includes the SeqA and SeqB proteins.

Choline kinase alpha 1 (ChoKα1) has recently become an interesting therapeutic target since its overexpression has been associated to tumorigenesis in many cancers. Nevertheless, little is known regarding hematological malignancies. In this manuscript, we investigated the effect of a novel and selective ChoKα inhibitor EB-3D in T acute lymphoblastic leukemia (T-ALL).The effect of EB-3D was evaluated in a panel of T-leukemia cell lines and ex-vivo primary cultures derived from pediatric T-ALL patients. We also evaluated in detail, using Reverse Phase Protein Array (RPPA), protein phosphorylation level changes in T-ALL cells upon treatment.The drug exhibits a potent antiproliferative activity in a panel of T-leukemia cell lines and primary cultures of pediatric patients. Moreover, the drug strongly induces apoptosis and more importantly it enhanced T-leukemia cell sensitivity to chemotherapeutic agents, such as dexamethasone and l-asparaginase. In addition, the compound induces an early activation of AMPK, the main regulator of cellular energy homeostasis, by its phosphorylation at residue T712 of catalytic subunit α, and thus repressing mTORC1 pathway, as shown by mTOR S2448 dephosphorylation. The inhibition of mTOR in turn affects the activity of several known downstream targets, such as 4E-BP1, p70S6K, S6 Ribosomal Protein and GSK3 that ultimately may lead to a reduction of protein synthesis and cell death.Taken together, our findings suggest that targeting ChoKα may be an interesting option for treating T-ALL and that EB-3D could represent a valuable therapeutic tool.

The effects of antisense oligonucleotides (ODNs) on nitric oxide (NO) production induced by lipopolysaccharide (LPS) were investigated using thioglycollate-induced mouse peritoneal macrophages. Antisense phosphorothioate ODNs (S-oligo) corresponding to a sequence in the neighborhood of the AUG initiation codon of a mouse inducible nitric oxide synthase (iNOS) mRNA, which has a G-quartet motif in its antisense sequence, inhibited NO induction in a dose-dependent manner. Antisense phosphodiester ODNs (D-oligo), 5'- and 3'-terminal phosphorothioate-modified antisense ODNs and control scramble and missense S-oligos had no such effect. In addition, control nonsense and two mismatched S-oligos, which include G-quartet motif in their sequences, inhibited NO induction to approximately 50% of those in the control. Antisense S-oligo showed the inhibitory effect on NO production by exposure of macrophages to various concentrations of LPS. Western blot analysis using anti-mouse inducible nitric oxide synthase (iNOS) antibody revealed that antisense S-oligo specifically removed an immunoreactive band at 130 kDa. In addition, the results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effect originated from a specific reduction of the targeted iNOS mRNA by hybridization with the antisense S-oligo. Furthermore, no ODNs affected beta-actin mRNA and tumor necrosis factor alpha (TNF-alpha) expression in macrophages stimulated by LPS. These findings demonstrated that antisense S-oligo inhibited NO production derived from iNOS expression in macrophages by an antisense mechanism, including the aptameric effect partially mediated by the G-quartet motif.

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.

Photoaffinity labeling of cholinephosphotransferase from rat liver microsomes directly by its substrate, [32P]CDP-choline or by a synthetic photoreactive CDP-choline analog, 3'(2')-O-(4-benzoyl)benzoyl [32P]CDP-choline (BB-[32P]CDP-choline), was examined for the possible identification of its molecular form on subsequent SDS-PAGE followed by 32P-autoradiography. When the partially purified cholinephosphotransferase was photoirradiated in the presence of [32P]CDP-choline, a considerable amount of 32P-radioactivity was incorporated into the TCA-insoluble component. This incorporation was dependent on irradiation time, Mg2+ or Mn(2+)-requiring and inhibited strongly by the presence of Ca2+. Either CDP-choline or CDP-ethanolamine inhibited the ultraviolet irradiation-dependent incorporation of 32P-radioactivity into the TCA-insoluble component in a dose-dependent manner, whereas neither phosphocholine or 5'-CDP had any effect on this process. These results strongly suggested that the observed 32P-incorporation from [32P]CDP-choline into the protein component could be a consequence of the covalent interaction between cholinephosphotransferase and its substrate, [32P]CDP-choline. Two polypeptides, 25 kDa and 18 kDa, with high 32P-radioactivity were clearly identified on a SDS gel after the direct photoaffinity labeling with [32P]CDP-choline for more than 5 min of ultraviolet irradiation. On the other hand, when BB-[32P]CDP-choline was used as a photoaffinity ligand, a single polypeptide with apparent molecular size of 55 kDa could be rapidly photolabeled within 2.5 min, then this band gradually lost its 32P-radioactivity with increasing time of ultraviolet irradiation. Thus, the overall results strongly indicated that cholinephosphotransferase in rat liver microsomes exists most likely as a 55 kDa polypeptide (or subunit) and that 25 kDa and 18 kDa peptides identified after the direct photoaffinity labeling with [32P]CDP-choline were probably the photo-cleavage products of cholinephosphotransferase during the prolonged ultraviolet irradiation, both of which could contain the catalytic domain of the original enzyme protein(s).

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.