Biomedical subjects
Publications and source records attributed to T Fukui.
Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.
We investigated the effect of prostaglandin E2 on the acquisition of cytokine-producing ability by naive CD4+ T cells in human cord blood. Naive CD4+ T cells were stimulated for 3 days with mouse monoclonal anti-CD3 Ab, then washed and expanded in IL-2-containing medium for 3 more days. These activated T cells produced IL-2, IL-4, IL-5, and IFN-gamma upon stimulation with PMA and ionomycin. PGE2 added at priming of naive T cells inhibited the production of IL-2 and IFN-gamma, but not of IL-4 and IL-5, in a dose-dependent manner. This change in the cytokine production profile induced by PGE2 was maintained in T cells restimulated with anti-CD3 in the absence of PGE2, expanded by IL-2, and stimulated with PMA and ionomycin. The mRNA expression of IFN-gamma and IL-2, but not that of IL-4, was also decreased in these cells. Forskolin and dibutyryl cAMP had a similar effect. PGE2 must exist at an early stage of T cell activation to inhibit priming for IL-2 and IFN-gamma production. PGE2 also showed this effect, even in the presence of exogenous IFN-gamma, at the primary stimulation. These results indicate that PGE2 inhibits the acquisition of the ability to produce IL-2 and IFN-gamma by acting directly on naive T cells. Our results suggest that PGE2 plays a role in facilitating the development of the Th2-type cytokine production profile.
Recent studies have shown that the NADPH oxidase participates in the generation of superoxide anion in non-phagocytic cells. Here we report the isolation and nucleotide sequence of a cDNA for the cytochrome b-558 alpha-subunit of the NADPH oxidase in rat vascular smooth muscle cells (VSMCs). The coding region of the cDNA was 93% homologous to mouse and 81% to human in nucleotide sequence and 96% homologous to mouse and 89% to human in the deduced amino acid sequence. Our results provide a tool with which to explore the mechanism of superoxide anion generation in rat VSMCs and other non-phagocytic cells.
The present study was undertaken on 10 patients with angina undergoing percutaneous transluminal coronary angioplasty. The angioplasty procedure consisted of two successive 30-second balloon inflations at 5 minute intervals. After the first inflation, nicorandil (0.1 mg/kg) was given intravenously over a 2-minute period. The second inflation was then performed 3 minutes after the completion of drug administration. Myocardial ischemia was measured as the magnitude of ST-segment elevation on the intracoronary electrocardiogram (intracoronary ECG) recorded from the guidewire. Nicorandil significantly reduced the magnitude of ST-segment elevation. Nicorandil did not change the heart rate-blood pressure product, nor the oxygen saturation of the blood sampled from the great cardiac vein, nor the velocity of coronary blood flow in those patients with no evidence of collaterals. These results favor the conclusion that nicorandil prolongs the intrinsic ability of cardiac myocyte to withstand oxygen deprivation. This salutary effect is possibly due to a direct cellular mechanism because nicorandil did not modify the peripheral and coronary hemodynamic parameters that govern myocardial oxygen consumption.
Crosslinking of the TcR-CD3 complex with immobilized anti-CD3 antibodies without sufficient co-stimulation induced cell death in human mature CD4+ T-cell lines. In these T cells, DNA fragmentation and morphological characteristics of apoptosis were seen. The anti-CD3-induced apoptosis was inhibited by co-culture with monocytes. The rescue signal provided by monocytes does not need to be present simultaneously with signals mediated by anti-CD3. When T cells were precultured with monocytes for 24 h before anti-CD3 stimulation and then the monocytes were removed from the culture, anti-CD3-induced T-cell apoptosis was also inhibited. To determine whether the monocyte-derived rescue signals were transduced by soluble factors or by direct cell-to-cell interaction with monocytes, we precultured T cells with monocytes separated by a micropore membrane which prevented T cell-monocyte physical interaction but not the diffusion of secreted molecules. In this system, rescue signals could not reach the T cells. To further assess the importance of physical interaction, we precultured T cells with fixed monocytes. T cells could not be rescued from apoptosis under these experimental conditions, either. The results considered collectively suggest that sufficient physical interaction with viable monocytes is important for the rescue of anti-CD3-induced apoptosis of CD4+ T cells.
Affinity labeling with nucleoside polyphosphopyridoxals, especially those with 3 or 4 phosphate groups, was effective for identifying the lysyl residue(s) located at or near the binding site for ATP, GTP, UDP-Glc, or ADP-Glc in various proteins. Furthermore, kinetic analysis of the mutant enzymes, in which the labeled lysyl residue was replaced with another amino acid by site-directed mutagenesis, provided evidence of its functional role. Affinity labeling of the mutant enzymes was useful for further identification of the hidden lysyl residue, which is unreactive in the wild-type enzyme but catalytically important. Comparison of the results of affinity labeling with different substrate analogues provided the information on the location of the labeled lysyl residue around the bound substrate. The affinity labeling reflected structural features of proteins, including their conformational flexibility.
In Japan, elderly patients who develop myasthenia gravis (MG) are increasing in number. However, there are few clinical reports concerning this issue. We evaluated the clinical manifestations, inducing or exacerbating factors, complications, treatments and prognosis of systemic MG in 11 patients older than 60 years of age. Bulbar symptoms were more frequent in these patients compared with younger MG patients, and 6 out of 11 cases (54.5%) were mistakenly diagnosed as cerebrovascular disorders. Among inducing or exacerbating factors of MG were psychological problems inherently involved with the aged, physical factors, and inappropriate termination or rejection of medication. Increase in the level of anti-Ach-R antibodies was recognized in 10 out of 11 cases (90.9%). A high percentage of the patients had thymoma (36.4%) and thyroid diseases (45.5%): 3 with Hashimoto's thyroiditis (27.3%), 1 with thyroid ophthalmopathy associated with hyperthyroidism, and 1 with simple goiter. Others were accompanied by ischemic heart disease, prostatic hypertrophy or stomach cancer. We treated these patients with corticosteroids, immunoglobulin, radiation for thymoma, or thymectomy in addition to administration of anticholinesterase agents. Prognostically, we found that duration of illness before death was shorter in those with onset later than 70 years of age. Seven out of 11 (63.6%) patients died of either aspiration pneumonia (4 cases), complications of thymectomy, congestive pulmonary edema or stomach cancer. There were no deaths associated with myasthenic crisis.
We investigated mirror movements observed in two patients skilled in playing the piano and compared these symptoms with those reported in patients associated with frontal lobe, corpus callosum or cervical cord lesion. We found the following common features in our two patients: 1) mirror movements were observed during skilled finger movements such as playing the piano, 2) these were observed in distal parts of the bilateral upper extremities, 3) contralateral imitative associated movements were seen concomitantly, 4) frontal lobe symptoms and callosal disconnection syndrome were not seen, 5) both patients recalled having mirror movements in their infancy, and one had family history. These characteristics of mirror movements in our patients were similar to those in patients associated with cervical cord lesion, but were different from those in patients associated with frontal lobe or corpus callosum lesion. It is suggested that abnormal pathways in the pyramidal tract or cervical cord lesion elicited mirror movements in our patients.
A 13-year-old boy presented with ophthalmoplegia, cerebella ataxia and areflexia subsequent to watery diarrhea and pyrexia. Campylobacter jejuni (PEN 2: LIO 4) was isolated from the stools. The patient had a high titer of IgG anti-GQ1b IgG antibody titers which decreased during the clinical course. Although C. jejuni has recently been recognized as the most common microorganism causing infection preceding Guillain-Barré syndrome, only six patients with Fisher's syndrome after this bacterial infection have been reported. This is the first report of a patient with Fisher's syndrome in whom C. jejuni was identified as a causative organism in Japan.
In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.
A binding protein which exhibits high affinity to [3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11 beta-HSD activity. These results show that the purified [3H]glycyrrhetinic-acid binding protein is 11 beta-HSD. From the molecular mass of 11 beta-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11 beta-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [3H]glycyrrhetinic acid binding and 11 beta-HSD activity indicate that the C30-carboxyl and C11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11 beta-HSD inhibition.
The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.
L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.
Stereoselective dehydrogenation of three isomers of trimethylsilylpropanol was carried out with horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1.) and optically active organosilicon compounds were obtained in a water-organic solvent two-layer system with coenzyme regeneration. Furthermore, we examined the effects of the silicon atom on stereoselectivity of HLADH compared to the corresponding carbon compounds. Substitution of the silicon atom for the carbon atom was found to improve the stereoselectivity of HLADH. For example, the optical purity of the remaining 1-trimethylsilyl-2-propanol was higher than 99% enantiomeric excess (ee) at 50% conversion, whereas that of the carbon analogue was 84% ee. This phenomenon was probably ascribable to the bulkiness of the organosilicon compounds derived from their longer Si-C bond. Kinetic analysis in an aqueous monolayer system demonstrated that the specific properties of the silicon atom greatly affected the reactivity of these substrate compounds.
The adherence of Escherichia coli and Pseudomonas aeruginosa to the epithelium of the gallbladders obtained from 32 patients with negative bile culture was quantified by a scanning electron microscope. Of the gallbladders, 5 were histologically normal (group A), 21 had chronic calculus cholecystitis (group B), and 6 had acute calculus cholecystitis (group C). The data were expressed as the mean +/- S.D. of the numbers of adherent bacteria to 1,000 microns2 of the gallbladder epithelium. The number of adherent E. coli were 0.1 +/- 0.2 in group A, 4.2 +/- 2.8 in group B, and 9.2 +/- 3.3 in group C. A similar result was also observed with P. aeruginosa. The number of adherent bacteria, both of E. coli and P. aeruginosa were significantly higher in group C than in groups A and B, and were also significantly higher in group B compared to group A. The amount of bacterial adherence paralleled that of the degree of epithelial damage, and the normal epithelium proved to have an inhibiting ability. Thus, a secondary bacterial infection is more likely to happen in patients with contaminated bile, and therefore, the treatment for acute cholecystitis should be based either on the results of a bile culture or according to predictive factors for bactibilia.
Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.
The present study was designed to test two hypotheses: (1) that angiotensin II (Ang II) stimulates endothelin-1 secretion in cultured rat mesangial cells and (2) that atrial and brain natriuretic peptides (ANP and BNP) inhibit the above-mentioned secretion in these cells. Ang II stimulated immunoreactive (ir) endothelin-1 secretion in a concentration-dependent manner between 10(-8) M and 10(-7) M. The protein kinase C (PKC) inhibitors from two chemical classes, H7 and staurosporine, inhibited secretion following such stimulation. The stimulatory effect of Ang II was also abolished in the PKC-depleted cells. Rat ANP(1-28) and rat BNP-45, which are the respective major circulating forms of ANP and BNP in rats, potently inhibited Ang II-stimulated endothelin-1 secretion in a concentration-dependent manner. Inhibition by ANP and BNP of Ang II-stimulated endothelin-1 secretion was paralleled by an increase in the cellular level of cyclic guanosine 5'-monophosphate (GMP). The addition of a cyclic GMP analogue, 8-bromo cyclic GMP, reduced the stimulated endothelin-1 secretion. Rat ANP(5-25) was less effective that rat ANP(1-28) with respect to inhibiting ir-endothelin-1 secretion and increasing cellular cyclic GMP. These findings indicate that Ang II stimulates endothelin-1 secretion in cultured rat mesangial cells by a mechanism probably involving activation of PKC, and that rat ANP and BNP inhibit this stimulated secretion through a cyclic GMP-dependent process.
We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.