Search PubMed⌕ Search

Biomedical subjects

M Ikeda

Publications and source records attributed to M Ikeda.

At least 775 records · Page 43Linked to original sources
PubMed1996

Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells.

We investigated the effects of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in the accumulation of nitrite, a stable metabolite of NO. Although cilostazol itself showed no effect on nitrite accumulation, it stimulated interleukin-1 beta-induced nitrite accumulation in a concentration-dependent manner (10(-8)-10(-5) M). This effect of cilostazol was completely abolished in the presence of NG-monomethyl-L-arginine, actinomycin D or dexamethasone. The cilostazol-induced nitrite production was accompanied by increased inducible NO synthase protein expression. In the presence of dibutyryl-cAMP, interleukin-1 beta-induced nitrite accumulation was further increased, but the stimulatory effect of cilostazol on nitrite accumulation was blunted. The effect of cilostazol was also abolished in the presence of Rp-8-bromoadenosine-3',5-cyclic monophosphorothioate, a competitive inhibitor of protein kinase A. Addition of cilostazol to the cultures significantly increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that cilostazol increases NO synthesis in interleukin-1 beta-stimulated smooth muscle cells, at least partially through a cAMP-dependent pathway.

Animals↗
PubMed1996

Long-term human T-cell culture system supporting hepatitis C virus replication.

We recently found that human T-cell leukemia virus type I-infected cloned MT-2C cells could support the replication of hepatitis C virus (HCV) up to 30 days postinoculation (p.i.). To obtain a more persistent HCV replication system using MT-2C cells, we examined the effect of various culture conditions after virus inoculation. We found that persistent HCV infection (at least 80 days) could be reproducibly achieved by reducing the temperature from 37 degrees C to 32 degrees C. Under this culture condition, HCV RNA could be detected in the cells up to 198 days p.i. Sequence analysis of HCV hypervariable region 1 revealed that certain HCV species became predominant during the culture period. We also demonstrated that virus transmission by the cell-free mode could be successfully repeated at least 4 times at 32 degrees C as opposed to only twice at 37 degrees C. This HCV-infected culture system will be useful for the various biological studies, including investigations into the mechanisms of HCV replication and multiplication.

Amino Acid Sequence↗
PubMed1996

Mutations of E2F-4 trinucleotide repeats in colorectal cancer with microsatellite instability.

Genetic instability at microsatellites in some colorectal cancer (CRC) have been linked to the defects of human mismatch repair genes, but the targets of these defective genes have been largely unknown. We screened CRC specimens for alteration of E2F-4 gene by analyzing both cDNA and genomic sequences along with replication error (RER+) phenotype. Two out of 20 sporadic CRC patients showed RER+ phenotype. We found tumor-specific copy number alteration in 13 consecutive trinucleotide (CAG) repeats within the coding exon of E2F-4 exclusively in these 2 specimens. Thus, E2F-4 may be a clue of the target gene of defective repair genes in CRC with genetic instability in addition to the TGF-beta type II receptor gene.

Base Sequence↗
PubMed1996

Site-specific modification of interleukin-2 by the combined use of genetic engineering techniques and transglutaminase.

Exploring a new method for the site-specific incorporation of functional groups into proteins, we have studied the combined use of genetic engineering techniques and enzymatic methods. Specifically, a short peptide for use as a substrate of guinea pig liver transglutaminase (TGase) is introduced at the N terminus of human interleukin-2 (hIL-2). The expressed chimeric protein (rTG1-IL-2) is chemically modified at a glutamine site in the appended sequence by TGase-catalyzed transamination with two amines, monodansylcadaverine (MDC), or a constructed derivative of poly (oxyethylene) (POE3). For the TGase-catalyzed modifications with MDC and POE3, 1 mol of donor was incorporated per mole of rTG1-IL-2, respectively. N-Terminal sequence analysis of MDC-modified rTG1-IL-2 (MDC-rTG1-IL-2) showed that the Gln-4 residue in the chimeric protein was site specifically modified with MDC. On the other hand, tryptic mapping of POE3-modified rTG1-IL-2 (POE3-rTG1-IL-2) by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS) suggested that one of the Gln sites in the appended sequence was modified with POE3. The POE3-rTG1-IL-2 retained full bioactivity relative to the unmodified molecule and rhIL-2. This methodology could be a new and general route for the site-specific modification of proteins.

Amino Acid Sequence↗
PubMed1996

Different DNA elements can modulate the conformation of thyroid hormone receptor heterodimer and its transcriptional activity.

Thyroid-hormone receptors (TRs) form heterodimers with retinoid-X receptors (RXRs) on thyroid-hormone-response elements (TREs). However, it is not known whether the formation of liganded TR/RXR heterodimer on a TRE alone is sufficient to dictate transcriptional activity. We designed several mutated DR4s (half-sites arranged as direct repeats with a nucleotide gap of 4) that bound TR/RXR heterodimers preferentially, and employed them to characterize functional and biochemical properties of the heterodimers on DNA. Although TR/RXR heterodimer binding was similar on some of the mutated DR4s, transient transfection assays showed that TRalpha failed to support triiodothyronine (T3)-stimulated transcription on "inactive" DR4s but mediated basal repression on both "active" and inactive mutated DR4. T3 binding assays showed that the mutated DR4s did not affect T3 binding to the heterodimer. Finally, partial proteolysis studies revealed that binding of active DR4 elements and T3 to the heterodimer synergistically enhanced heterodimerization-induced protease resistance of TR, but not RXR, in the heterodimer. These results suggest that: 1) liganded TR/RXR heterodimer binding to a DR4 is not sufficient for transcriptional activation of the target gene, and 2) DNA sequences in specific TREs may modify T3-mediated transcription by affecting the conformation of the liganded heterodimer.

Binding Sites↗
PubMed1996

Post-translational modifications of Ras and Ral are important for the action of Ral GDP dissociation stimulator.

Ral GDP dissociation stimulator (RalGDS) is a GDP/GTP exchange protein of Ral and a new effector protein of Ras. Therefore, there may be a new signaling pathway from Ras to Ral. In this paper, we examined the roles of the post-translational modifications of Ras and Ral on this new signal transduction pathway. The post-translationally modified form of Ras bound to RalGDS more effectively than the unmodified form. The modification of Ras was required to regulate the distribution of RalGDS between the cytosol and membrane fractions in COS cells. The post-translational modification of Ral enhanced the activities of RalGDS to stimulate the dissociation of GDP from and the binding of GTP to Ral. Furthermore, the modified form of Ral bound to Ral-binding protein 1 (RalBP1), a putative effector protein of Ral, more effectively than the unmodified form. Taken together with the observations that Ras and Ral are localized to the membranes, these results suggest that the post-translational modifications of Ras and Ral play a role for transmitting the signal effectively on the membranes in the signal transduction pathway of Ras/RalGDS/Ral/RalBP1.

Amino Acid Sequence↗
PubMed1996

Vitamin B12 amplifies circadian phase shifts induced by a light pulse in rats.

Vitamin B12 (VB12) is a putative modulator of the human circadian clock, improving entrainability to the 24 h light-dark cycle. The present study was intended to elucidate the mechanism of VB12 action in an animal model. In male rats free-running under constant dim illumination, a single light pulse of 50-1000 lux for 20 min given at circadian time (CT) 20 induced a 0.28 to 1.08 h phase advance and at CT 14 induced a 0.54 to 2.10 h phase delay. A 3 h intracerebroventricular (icv) infusion of 30 nmol VB12 starting 2 h prior to a 20 min 200 lux light pulse significantly amplified phase shifts in comparison with saline-treated or untreated controls. The mean phase advance (1.13 h) was 1.8-fold greater than that of saline-infused controls, whereas the mean phase delay (2.28 h) was 2.9-fold greater. These values were comparable to the maximal phase shifts caused by 1000 lux light pulses in untreated rats. Since the same VB12 treatment alone had failed to induce a phase shift in a previous experiment, these results indicate that VB12 strongly enhanced light pulse-induced phase shifts and thus augmented the entrainability of the circadian clock to light.

Animals↗
PubMed1996

Transcriptional activation of RACTK1 K+ channel gene by apical alkalization in renal cortical collecting duct cells.

We have previously demonstrated that RACTK1 cDNA encodes a pH sensitive K+ channel expressed in the apical side of renal collecting tubule cells. To determine whether extracellular pH induces the RACTK1 gene expression in the renal cortical collecting duct (CCD) cells, we measured mRNA of the RACTK1 using cultured rabbit CCD cells. Alkalization of incubation medium activated the transcription of the RACKTK1 gene in a time- and dose-dependent manner after 1 h, and reached a maximal level after 12 h. To examine whether the stimulation of mRNA by alkalization of body fluid occurs also in vivo, mRNA levels were measured in mice loaded with acid or alkali. The RACTK1 mRNA was increased in association with the rise in urinary pH. To examine side face of the effect of pH on stimulation of mRNA, we observed the effect of pH in the apical or the basolateral side in the preparation where CCD cells were cultured on filter membrane supports. Alkalization of the apical side but not of the basolateral side, was shown to be a determinant in inducting the RACTK1 mRNA. These findings suggest that, in addition to rapid direct regulation of RACTK1 K+ channel conductance by intracellular pH, this channel is also regulated by the changes in luminal pH through synthesis of channel protein by transcriptional activation.

Animals↗
PubMed1996

The differences in Ca(2+)-sensitivity of protein kinase C in platelets from Wistar Kyoto rat and stroke-prone spontaneously hypertensive rat.

Thrombin-induced phosphorylation of 47 kDa protein (P47) in platelets, a substrate of protein kinase C (PKC), was defective in stroke-prone spontaneously hypertensive rats (SHRSP) (Hypertens. 14, 304-315, 1989). Platelet PKC from SHRSP and Wistar Kyoto rats (WKY) was partially purified and Ca(2+)-sensitivity of PKC activity was examined to understand the defect in the protein phosphorylation. When the platelets from SHRSP and WKY were homogenized in a buffer containing 10 mM EGTA and 2 mM EDTA, approx. 80% of PKC was in the cytosol fraction. PKC in this fraction was purified by DE52 and hydroxyapatite column chromatography. Both platelet PKC preparations contained only PKC-alpha, and there was no significant difference in the Ca(2+)-dependency of activity between them. When the platelets from SHRSP and WKY were homogenized in a buffer containing 10 microM CaCl2, 90% of PKC was found to be bound to the membrane. PKC was extracted from the membrane with a buffer containing 2.5 mM EGTA and 2.5 mM EDTA, and purified by DE52 column chromatography. PKC from WKY platelets eluted as a single peak whereas that from SHRSP platelets eluted as two peaks (peak 1 and peak 2). Ca(2+)-sensitivity of peak 1 PKC was much lower than that of WKY PKC. In contrast, the Ca(2+)-sensitivity of peak 2 PKC appeared to be slightly higher than that of WKY PKC. The specific activity of peak 2 PKC was 4% to 5% of that of peak 1 and WKY PKC. These results suggest that there are two different types of PKC, normal and low Ca(2+)-sensitive in SHRSP platelets. Defective P47 phosphorylation in SHRSP platelets might be attributable to the occurrence of this low Ca(2+)-sensitive PKC.

Animals↗
PubMed1996

[The association of self-rated health and mortality--a 7-year follow-up study of a Japanese community].

This study aims to examine whether self-rated health is an independent predictor of cause-specific mortality even after the influence by a variety of factors in relation to mortality reported by previous studies have been excluded. This study included randomly selected 4,259 inhabitants (1,827 men and 2,432 women) in Y city, Fukuoka prefecture, Japan, aged from 30 to 79 in 1987. These subjects were surveyed in 1987 by self-administered questionnaires regarding self-rated health as well as life habits, and followed up for their vital status and underlying causes of death. After excluding the individuals who were lost to follow up or who didn't respond to the question on self-rated health, 4,046 individuals were analysed to see the relationship between self-rated health and cause-specific mortality by Cox proportional hazard models, controlling for sex, age, smoking, BMI, medical care use and ADL. It was shown that relative risks for all causes, cancer, circulatory disease and other causes among the unhealthy group were 2.95 (95% CI: 1.93-4.50), 2.96 (1.53-5.73), 2.32 (0.86-6.26) and 4.09 (2.12-7.89), relatively. In the analyses of the subgroup (subjects without diseases in 1987 or subjects excluding deceased cases within first 3 years after follow-up), to avoid selection bias, the association between self-rated health and mortality was substantially similar to the results obtained in the former analysis, even the association was weakened. Even after excluding both of the subjects with diseases in 1987 and the subjects who died in the first 3 years after follow-up, self-rated health could be associated with mortality from all causes (RR = 1.89, 95%CI; 0.91-3.94). From the results it is suggested that self-rated health itself can be the independent predictor of mortality.

Adult↗
PubMed1996

Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.

We have recently reported that a novel molecule, murine IFN-gamma-inducing factor (IGIF) produced by mouse liver cells, possesses potent biologic activities, including the induction of IFN-gamma production by spleen cells and the enhancement of NK cell cytotoxicity. In this paper, we report on the isolation of human IGIF cDNA clones from normal human liver cDNA libraries using murine IGIF cDNA as a probe. The amino acid sequence deduced from the human cDNA clones indicated a 193-amino acid precursor peptide and revealed 65% homology with that of murine IGIF. The amino acid sequence of IGIF also included an IL-1 signature-like sequence. Subsequently, the cloned cDNA was expressed in Escherichia coli, and preliminary studies on the biologic activities of the recombinant protein were performed. The recombinant human IGIF induced IFN-gamma production by mitogen-stimulated PBMC and enhanced NK cell cytotoxicity, in a manner similar to murine IGIF. In addition, recombinant human IGIF also augmented granulocyte-macrophage-CSF production and decreased IL-10 production, but had no effect on IL-4 production by Con A-stimulated PBMC. Based on these pleiotropic effects of IGIF, we propose that this novel cytokine be designated as IL-18.

Adult↗
PubMed1996

Cadmium contents in rice samples from various areas in the world.

Rice samples consumed by local populations were collected in 17 areas in the world, mostly from Asia, i.e. ten areas, but eight areas outside of Asia were also included during the period of 1990 to 1995. The samples amounted to 1546 in total, and were analyzed for cadmium (Cd) by electrothermal atomic absorption spectrometry. The data show that the highest and the lowest geometric means of Cd contents in rice from Asia was 55.70 and 2.67 ng/g, respectively, and 133.20 and 0.88 ng/g outside of Asia. The geometric mean Cd contents in rice from Japan was 55.70 ng/g, which is essentially similar to the levels determined in the early 1980s. We conclude that a substantial difference exists in Cd contents in rice for local consumption depending on the areas in the world, and that there is no significant changes in Cd levels in rice harvested in Japan over the past 10 years.

Analysis of Variance↗
PubMed1996

Inhibition of cholesterylester accumulation by 17 beta-estradiol in macrophages through activation of neutral cholesterol esterase.

Premenopausal women are at a lower risk of coronary heart disease relative to age matched men. However, the underlying mechanisms are not clearly understood. This article studies the effects of 17 beta-estradiol (17 beta-E2) at physiological concentrations on the cholesterylester metabolism in macrophages (J774 A.1 cells) with a particular focus on neutral cholesterol esterase (N-CEase). Cells were incubated with beta-VLDL, [1-14C]oleic acid and 17 beta-E2 (0.25 and 2.5 nM). 17 beta-E2 dose-dependently reduced cholesteryl-[1-14C]oleate (14C-CO) at 36 h and 48 h relative to the control. It also stimulated hydrolysis of 14C-CO in foam cells on 36 h and 48 h incubation. In addition, 17 beta-E2 markedly increased N-CEase activity at 24 h and 36 h. This increase preceded the enhanced hydrolysis of cholesterylester, 17 alpha-E2 (inactive isomer), estrone and estriol had no stimulatory action on N-CEase, whereas progesterone and testosterone inhibited it. 17 beta-E2-treatment (24 h) increased the activity of cyclic AMP-dependent protein kinase (A-kinase). DEAE-cellulose column chromatography revealed that an isoform (type II) of A-kinase appeared in 17 beta-E2-treated cells in addition to type I of A-kinase found in the control cells. These results suggest that inhibition of cholesterylester accumulation in macrophages by 17 beta-E2 is mediated by an enhancement of N-CEase activity possibly through an increase in A-kinase.

Animals↗
PubMed1996

CTBP1/RBP1, a Saccharomyces cerevisiae protein which binds to T-rich single-stranded DNA containing the 11-bp core sequence of autonomously replicating sequence, is a poly(deoxypyrimidine)-binding protein.

South-Western screening of a glutathione-S-transferase fusion protein library constructed from the yeast Saccharomyces cerevisiae genomic DNA lead to isolation of core T-rich-strand-binding protein (CTBP) clones that bound to single-stranded DNA containing the T-rich-strand of the 11-bp core sequence of autonomously replicating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA-binding and single-stranded DNA-binding protein of S. cerevisiae. GST-CTBP1 as well as the full-length fusion protein with RBP1 (GST-RBP1) bind exclusively to the T-rich strand of the core sequence with an apparent dissociation constant of 5 nM, but not to the A-rich strand or double strand of the same sequence. Mutations within the core which reduce the number of T or C residues decrease the affinity of this protein. In keeping with this, binding of GST-CTBP1 to the core sequence is efficiently completed by poly(dT), poly(dT-dC) or poly(dC), but not by poly(dA) or poly(dG) to significant extents. Among polyribonucleic acids, GST-CTBP1 binds to poly(U) and poly(I) with greatest affinity, whereas GST-RBP1 binds to RNA in a rather non-specific manner. In no cases was affinity for RNA greater than that for DNA. Our results indicate that CTBP1/RBP1 is a polydeoxypyrimidine-binding protein of S. cerevisiae. CTBP1 contains two sets of an RNA-recognition motif (RRM) and a glutamine stretch. The binding affinity of the N-terminal or C-terminal set containing one RRM and one glutamine stretch is nearly two orders of magnitude lower than that of the wild-type CTBP1 containing both sets. The isolated N-terminal or C-terminal RRM alone (RRM1 and RRM2, respectively) is sufficient for binding nucleic acids with the binding specificity similar to that of the wild-type RRM, although the binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that the two RRMs present in CTBP1/RBP1 have differential binding affinities and that the high affinity of RRM for polydeoxypyrimidine results from synergy between two lower-affinity RRMs.

Alcohol Oxidoreductases↗
PubMed1996

Left-right asymmetric expression of the TGF beta-family member lefty in mouse embryos.

Examples of lateral asymmetry are often found in vertebrates, such as the heart being on the left side, but the molecular mechanism governing the establishment of this left-right (L-R) handedness is unknown. A diffusible morphogen may determine L-R polarity, but a likely molecule has not so far been identified. Here we report on the gene lefty, a member of the transforming growth factor-beta family, which may encode a morphogen for L-R determination. Lefty protein contains the cysteine-knot motif characteristic of this superfamily and is secreted as a processed form of relative molecular mass 25K-32K. Surprisingly, lefty is expressed in the left half of gastrulating mouse embryos. This asymmetric expression is very transient and occurs just before the first sign of lateral asymmetry appears. In the mouse mutants iv and inv, which cause situs inversus, the sites of lefty expression are inverted, indicating that lefty is downstream of iv and inv. These results suggest that lefty may be involved in setting up L-R asymmetry in the organ systems of mammals.

3T3 Cells↗
PubMed1996

Purkinje cells express neuronal nitric oxide synthase after methylmercury administration.

To study the effects of chemical injury on the cerebellar nitric oxide synthase (NOS), we administered methylmercury chloride subcutaneously to mice, 10 mg/kg/day for 9 days. In the methylmercury-treated cerebellum. Purkinje cells were positive both for NADPH-diaphorase and for neuronal NOS. Calcium-dependent NOS activity was increased to 160% of the controls. The present study suggests the ability of Purkinje cells to produce NO through the expression of neuronal NOS.

Animals↗
PubMed1996

Hemostasis activation during sclerotherapy of lower extremity varices.

The influence of compression sclerotherapy upon hemostasis activation was investigated in 41 consecutive patients with lower extremity varices by serial measurement of thrombin-antithrombin III complexes (TAT), D-dimer, fibrinogen and C-reactive protein (CRP). Blood sampling was carried out before operation and on the 7th and 28th post-operative day in patients randomly assigned to either the control group (n = 18), in which high ligation of sapheno-femoral junction and local excision of varices were performed, or the sclerotherapy group (n = 23) in which the comparable surgical intervention and compression sclerotherapy using hypertonic saline were performed simultaneously. In both groups, the TAT, D-dimer and fibrinogen concentrations at day 7 were significantly elevated compared to the value before operation while CRP showed no significant change during the observation period. In the sclerotherapy group, higher incidence of superficial thrombosis was observed and the TAT concentration at day 7 was significantly higher than that in the control group (p < 0.01), and the TAT at day 28 was still significantly elevated compared to the pre-operative level (p < 0.05). However, no relationship between TAT and D-dimer concentrations and the extent of superficial thrombosis was observed. We conclude that compression sclerotherapy for lower extremity varices causes latent activation of coagulation system and can be a risk factor for venous thromboembolism.

Antithrombin III↗