Biomedical subjects

M Ui

Publications and source records attributed to M Ui.

At least 55 records · Page 3Linked to original sources

PubMed1999

Fra-2-positive autoregulatory loop triggered by mitogen-activated protein kinase (MAPK) and Fra-2 phosphorylation sites by MAPK.

We reported previously that activation of endogenous activator protein 1 (AP-1) in chicken embryo fibroblasts is essential for the cellular transformation induced by v-src, and we further showed that the activation of AP-1 is accompanied by elevation of Fra-2 and c-Jun expression and also high-level phosphorylation of Fra-2 by activated endogenous extracellular signal-regulated kinase [mitogen-activated protein kinase (MAPK)]. Here, we report that the transcriptional activity of Fra-2/c-Jun heterodimer was greatly enhanced by cotransfecting a constitutively active mutant of MEK1 gene (MEK-DD) into F9 cells, indicating that Fra-2 was converted into an active transactivator after phosphorylation by MAPK. High-level expression of MEK-DD alone was sufficient to induce clear cellular transformation of chicken embryo fibroblasts, which caused constitutive activation of endogenous MAPK, hyperphosphorylation of Fra-2, and elevation of fra-2 and c-jun gene expression. These results indicate that phosphorylation of Fra-2 by MAPK plays an important role in stimulating endogenous AP-1 activity in a positive autoregulation mechanism, in which phosphorylated Fra-2 induces fra-2 expression through AP-1 binding sites present in its promoter. We also localized the Fra-2 phosphorylation sites by MAPK to three threonine and three serine residues in the COOH-terminal region by means of site-directed mutagenesis and showed that the threonine residues were more susceptible to MAPK.

Amino Acid Sequence↗

PubMed1998

Downregulation of mRNA expression of Edg-3, a putative sphingosine 1-phosphate receptor coupled to Ca2+ signaling, during differentiation of HL-60 leukemia cells.

We measured the mRNA expression of the recently identified putative sphingosine 1-phosphate (S1P) receptors, i.e., Edg-1, AGR16/H218, and Edg-3, in HL-60 leukemia cells. Of these putative receptors, Edg-3 mRNA was abundantly expressed in undifferentiated HL-60 cells. Further, its mRNA expression was markedly downregulated by inducers of cell differentiation such as dibutyryl cAMP, retinoic acid, and 1alpha, 25-dihydroxyvitamin D3. The reduction of mRNA expression was associated with the attenuation of an S1P-induced increase in cytoplasmic free Ca2+ concentration. Thus, Edg-3, whose mRNA expression is downregulated during cell differentiation, may be responsible for the S1P-induced Ca2+ response in HL-60 leukemia cells.

3T3 Cells↗

PubMed1998

Stimulatory and inhibitory actions of lysophosphatidylcholine, depending on its fatty acid residue, on the phospholipase C/Ca2+ system in HL-60 leukaemia cells.

We examined the mechanism of action of lysophosphatidylcholine (LPC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflammatory disorders, in HL-60 leukaemia cells. Extracellular 1-palmitoyl LPC increased the intracellular Ca2+ concentration in association with production of inositol phosphate. These actions of LPC were markedly inhibited by treatment of the cells with pertussis toxin and U73122, a phospholipase C inhibitor. The lipid-induced stimulation of the phospholipase C/Ca2+ system was also attenuated in the dibutyryl cAMP-induced differentiated (neutrophil-like) cells, in which phospholipase C activation induced by NaF or formyl-Met-Leu-Phe was enhanced. In contrast with the stimulatory action of 1-palmitoyl LPC, 1-stearoyl LPC was inhibitory for the phospholipase C/Ca2+ system stimulated by NaF as well as by 1-palmitoyl LPC or other Ca2+-mobilizing agonists. In a cell-free system, only an inhibitory effect on phospholipase C activity was observed even by 1-palmitoyl LPC; 1-stearoyl LPC was more inhibitive than 1-palmitoyl LPC. Taken together, these results suggest that atherogenic and inflammatory LPC exerts both stimulatory and inhibitory actions on the phospholipase C/Ca2+ system depending on the species of fatty acid residue of the lipid; the stimulatory effect is possibly mediated through G-protein-coupled receptors; the inhibitory effect might be caused by dysfunction of the components involved in the enzyme system owing to the amphiphilic nature of the lipid. 1-Palmitoyl LPC prefers the former receptor stimulation at least in intact cells, but 1-stearoyl LPC preferentially exerts the latter inhibitory action.

Bucladesine↗

PubMed1998

Two proteins translated by alternative usage of initiation codons in mRNA encoding a JunD transcriptional regulator.

The junD gene encodes one component of the transcription factor, AP-1. Since two forms of JunD protein have been reported, we analyzed here the molecular mechanisms involved in the isoform production. Immunochemical analysis indicated that the longer and shorter forms of mouse JunD (JunD-L and JunD-S, with apparent molecular weights of 44 and 39 kDa, respectively) differ in their content of an N-terminal peptide. Mutational analysis further indicated that JunD-S is the translational product initiated at the third AUG located 144 bp from the first AUG, at which JunD-L translation starts. Such production of two junD isoforms from a single mRNA using the same reading frame seems to be conserved in human, rat, and chicken. To examine the functional differences between the isoforms, each type of JunD was exclusively expressed by the use of retrovirus vectors harboring the mutated junD gene. The exogenous expression of either one of these forms did not cause cellular transformation of NIH3T3, but suppressed the anchorage-independent growth of NIH3T3 transfor-bold by the activated K-ras or v-src gene. These two isoforms were expressed in all the mouse tissues examined and in various cell lines established from human tumors, though the expression ratio between JunD-L and JunD-S varied, suggesting that some factor(s) modulate the alternative usage of the initiation codon of the junD gene.

Amino Acid Sequence↗

PubMed1998

Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1.

Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1alpha (EF1alpha), was first described to be essential for the G1 to S phase transition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1alpha-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.

Amino Acid Sequence↗

PubMed1998

Biochemical and functional analysis of highly phosphorylated forms of c-Jun protein.

We report here that, upon UV irradiation or growth stimulation, endogenous c-Jun (40 kDa) in chicken embryo fibroblasts (CEF) is converted into several forms with apparently higher molecular weights in SDS-polyacrylamide gel electrophoresis (45, 44, 42 kDa). Two of the bands (44 and 45 kDa) were transient after growth stimulation, but were much more persistent after UV irradiation. In both cases, the drastic mobility shifts were accompanied with the activation of endogenous JNK activity but not of MAPK activity, and the bands were shown to represent different phosphorylation states of c-Jun rather than ubiquitinated c-Jun. Biochemical analysis indicated that phosphorylation at Ser63 and Ser73 was not sufficient to produce these drastic mobility shifts, which additionally required phosphorylation at Thr91 and Thr93. Substitution of both Ser63 and Ser73 with either Ala or Asp had no significant effect on the transforming activity of c-Jun, but the mutants failed to show drastic mobility shifts even after UV irradiation. These results indicate that Ser63 and Ser73 are essential for the drastic mobility shifts and further suggest that the highly phosphorylated forms of c-Jun are not directly involved in cellular transformation.

Amino Acid Sequence↗

PubMed1998

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. Role of cytoplasmic tail phosphorylation in each process.

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.

Amino Acid Sequence↗

PubMed1998

Differential routes of Ca2+ influx in Swiss 3T3 fibroblasts in response to receptor stimulation.

Ca2+ influx into cells in response to stimulation of various receptors was studied with Swiss 3T3 fibroblasts. The mechanisms involved were found to be so diverse that they were classified into four groups, Type I to IV. Type-I influx occurred, via pertussis toxin-susceptible G-proteins, immediately after internal Ca2+ mobilization by bradykinin, thrombin, endothelin, vasopressin or angiotensin II. Type-II influx induced by bombesin differed from Type I in its insusceptibility to pertussis toxin treatment. Ca2+ influx induced by prostaglandin E1, referred to as Type-III influx, was unique in that phospholipase C was apparently not activated without extracellular Ca2+, strongly suggesting that the Ca2+ influx preceded and was responsible for InsP3 generation and internal Ca2+ mobilization. More Ca2+ entered the cells more slowly via the Type-IV route opened by platelet-derived and other growth factors. These types of Ca2+ influx could be differentiated by their different susceptibilities to protein kinase C maximally activated by 1 h of exposure of cells to PMA, which inhibited phospholipase Cbeta coupled to receptors involved in Type-I and -II influx but did not inhibit growth-factor-receptor-coupled phospholipase Cgamma. Type-I and -II Ca2+ influxes, together with store-operated influx induced by thapsigargin, were not directly inhibited by exposure of cells to PMA, but Type-III and -IV influxes were completely inhibited. In addition, stimulation of receptors involved in Type-I and -IV Ca2+ influx, but not Type-II and -III influx, led to phospholipase A2 activation in the presence of extracellular Ca2+. Inhibition of Type-I and -IV Ca2+ influxes by their respective inhibitors, diltiazem and nifedipine, resulted in abolition of phospholipase A2 activation induced by the respective receptor agonists, in agreement with the notion that Ca2+ influx via these routes is responsible for receptor-mediated phospholipase A2 activation.

3T3 Cells↗

PubMed1998

Differential mitogenic actions of alpha 1- and beta-adrenergic agonists on rat hepatocytes.

alpha 1-Adrenergic receptor-mediated responses are overwhelming in adult rat hepatocytes. Inversely, beta-responses are predominant over alpha 1-responses in the hepatocytes that have been cultured at a low cell density (10(4) cells/cm2) for 24 h. The insulin-EGF-induced DNA synthesis in the beta-response-dominant hepatocytes was doubled by beta-agonists or cAMP-generating agents added far behind (16-20 h) the addition of insulin/EGF; i.e., immediately before the entry into the S-phase of the cell cycle. Agonists of alpha 1-adrenergic or other Ca2+, mobilising receptors added to the alpha 1-response-dominant hepatocytes increased DNA synthesis only if they were added within 1-2 h after the addition of insulin/EGF, at the early stage of G1-phase. Agonists of "non-dominant" receptors were rather antagonistic to agonists of "dominant" receptors. Thus, agonists of alpha 1-adrenergic (and other Ca2+ mobilising) receptors and agonists of beta-adrenergic (and other cAMP-generating) receptors acted as comitogens in their own particular manners in the presence of growth factors in hepatocytes in which the respective receptor functions were dominant.

Adrenergic alpha-Agonists↗

PubMed1998

Involvement of phosphoinositide 3-kinase in regulation of adhesive activity of highly metastatic hepatoma cells.

We have established hepatoma clones from benzopyrene-treated liver cells, one of which (G-5) shows extensive metastasis to the lung when injected subcutaneously into mice [Tanigaki, Y. et al. (1995) Invasion Metastasis 15, 70-80]. In the present study, we performed in vitro assays suitable for examination of the adhesive and invasive properties of the highly metastatic cells. G-5 cells efficiently entered the pores of fibronectin-coated filters. Treatment of the cells with an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), wortmannin, significantly impaired the invasive activity. A structurally unrelated inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also prevented invasion. Both inhibitors suppressed cell adhesion to fibronectin-coated dishes. G-5 cells were next transfected with a mutant regulatory subunit (Deltap85) of PI 3-kinase, which was expected to impair the function of PI 3-kinase. The transfectants showed suppressed adhesion to the dishes and did not efficiently migrate into the filters. The lower adhesive ability of the transfected cells was not further affected by inhibitors of PI 3-kinase. Thus, PI 3-kinase activity contributes significantly to the adhesive and invasive properties of G-5 cells.

Androstadienes↗

PubMed1998

Infectivity and immunogenicity of SIVmac/HIV-1 chimeric viruses (SHIVs) with deletions in two or three genes (vpr, nef and vpx).

Two SHIVs with two or three genes deleted (SHIV-drn and SHIV-dxrn) were constructed. The inoculation of monkeys with SHIV-drn resulted in short-term viremia, but inoculation with SHIV-dxrn did not. At 68 weeks post-inoculation, the monkeys were reinoculated with a 100-fold higher dose of each SHIV, but none showed viremia. Killer cell activities against HIV-1 Env were detected in the SHIV-drn- and SHIV-dxrn-inoculated monkeys. Cross-reactive killer activity against HIV-1 Gag and SIVmac Gag was observed in one monkey. Antibodies were not detected in the SHIV-dxrn-inoculated monkeys, but the SHIV-drn-inoculated monkeys showed an anamnestic antibody reaction. These data indicate that SHIV-drn is infectious to and immuno-inducible in macaques but SHIV-dxrn is not.

AIDS Vaccines↗

PubMed1998

A new approach to AIDS research and prevention: the use of gene-mutated HIV-1/SIV chimeric viruses for anti-HIV-1 live-attenuated vaccines.

The lack of a suitable animal model is a major obstacle to developing anti-HIV-1 vaccines. We successfully generated an SIVmac/HIV-1 chimeric virus (SHIV) (designated as NM-3rN) that contains the HIV-1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM-3rN, we developed an evaluation system for anti-HIV-1 Env-targeted vaccines. For the purpose of making the vaccine, a series of gene-mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long-term anti-virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM-3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV-1 but also against an antigenically different HIV-1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV-MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene-deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide-range immunity against HIV-1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti-HIV-1 live vaccines for humans.

AIDS Vaccines↗

PubMed1998

Genomic analysis of the viral population in genital secretions early after infection of simian immunodeficiency viruses in macaque monkeys.

To clarify the change in the viral population during passage from the vaginal cavity to blood circulation and vice versa, we examined the viral clones detected in cells in vaginal washes (VWCs) early after inoculation and after systemic infection with polyclonal SIV. In two intravaginally inoculated monkeys, the viral clones found in VWCs at 18 days p.i. were shown to be some of those contained in the inoculum, whereas the viral population in the peripheral blood mononuclear cells (PBMCs) was a monotype. This gradual decrease of viral clones suggested the possible existence of two barriers, one at the genital tract and the other between the genital tract and the blood. Later, at one month p.i., the viral clones in VWCs became rather restricted, whereas those in PBMCs diverged from a single clone to several clones. This suggested that different mechanisms affect the viral populations in PBMCs and VWCs. In order to examine how the viral population was affected by passage from the blood to the vaginal cavity, a monkey was intravenously inoculated and the viral clones in VWCs were analyzed at 14 days p.i., at a time of the heterogeneous population in PBMCs. The viral population in VWCs was found to be a single clone and this clone was a minor type in PBMCs, suggesting that the major clone in PBMCs was not always secreted to the vaginal cavity.

Animals↗

PubMed1998

A new system for stringent, high-titer vesicular stomatitis virus G protein-pseudotyped retrovirus vector induction by introduction of Cre recombinase into stable prepackaging cell lines.

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the gag-pol genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neo(r)) gene and a poly(A) signal flanked by a tandem pair of loxP sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neo(r) expression. By inserting an mRNA-destabilizing signal into the 3' untranslated region of the Neo(r) gene to reduce the amount of Neo(r) transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding beta-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 x 10(6) infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.

Animals↗

PubMed1998

Microinjection of activated phosphatidylinositol-3 kinase induces process outgrowth in rat PC12 cells through the Rac-JNK signal transduction pathway.

We have previously shown that sustained phosphatidylinositol (PI)-3 kinase activity is necessary for neurite outgrowth of PC12 cells induced by nerve growth factor (NGF). Microinjection of a constitutively active mutant of PI-3 kinase induced process formation suggesting that PI-3 kinase is indeed involved in the neurite outgrowth. However, the processes appeared to be incomplete neurites as they had very poor organization of F-actin and GAP43 antigen. The microtubule network was enhanced in the process-bearing cells and process formation was inhibited by colchicine suggesting that microtubules play an important role in process formation downstream of PI-3 kinase. These cell responses were inhibited by dominant-negative mutants of Rac and Sek1/SAPK but not by a dominant-negative mutant Ras and PD98059, a MAP kinase kinase (MEK) inhibitor, suggesting that not the Ras-MAP kinase pathway but the Rac-Jun N-terminal kinase (JNK) pathway is involved in process formation.

Animals↗

PubMed1998

Rapid and progressive CD4+ decline in a monkey infected with an SIV+HIV-1 chimeric virus.

We previously constructed a simian immunodeficiency virus+human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4+ lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Transcriptional analysis of Marek's disease virus (MDV) genes in MDV-transformed lymphoblastoid cell lines without MDV-activated cells.

Spontaneously activated MDV is rarely included in MDV-transformed cells, while it may influence the results of transcriptional analysis. A population consisting of 10(3) MDV-transformed cells probably did not include spontaneously activated MDV, since the estimated frequency of MDV-transformed cells including activated MDV was below 0.01% according to limiting-dilution polymerase chain reaction (PCR) and the presence of the major early antigen pp38 in 6 transformed cell lines. Reverse transcriptase-PCR (RT-PCR) products corresponding to ICP27, pol, TK, US3, A41, gA, gB and UL50 genes were undetectable in 10(3) cells by Southern hybridization of the RT-PCR products. Transcripts of the VP16 and SORF2 genes were detected in the 10(3) cells of MSB-1, and the pp14 gene transcript was found in 10(3) cells of RPL-1 but not in 10(3) cells of HPRS-1, MOGA-2, MSB-1 or MTB-1. A transcript corresponding to the ICP4 sequence was detected as a 0.7 kbp RT-PCR product in 10(3) cells of these MDV cell lines but not in the retrovirus-transformed 1104B1 cell line. The transcript corresponding to the 0.7 kbp RT-PCR product suggested a splice by its size and sequence. Thus, transcriptional analysis of 10(3) MDV-transformed cells revealed that the transcript corresponding to the ICP4 sequence was a common transcript in latently infected MDV-transformed cells, while most of the genes did not transcribe in these cells.

Animals↗

PubMed1997

Exogenous sphingosine 1-phosphate induces neurite retraction possibly through a cell surface receptor in PC12 cells.

Exogenous sphingosine 1-phosphate (S1P), like lysophosphatidic acid (LPA), induced neurite retraction or cell rounding in differentiated PC12 cells. The lysosphingolipid-induced shape change was detected at as low as 1 nM; however, a significant accumulation of intracellular S1P was not detected until 1 microM S1P was applied. Moreover, although exogenous sphingosine caused a significant increase in intracellular S1P by sphingosine kinase-catalyzed phosphorylation, the effect on the shape change was marginal. Exposure of the cells to the immobilized S1P in which the lipid was covalently linked to a glass carrier also resulted in the shape change. These results suggest that the exogenous S1P-induced shape change does not require uptake of the lipid into the cells but possibly requires interaction with a cell surface receptor in the neuronal cells.

Animals↗