Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 37 records · Page 2Linked to original sources

PubMed1992

Holter ECG recordings before and after surgical treatment of hyperthyroidism: circadian pattern of heart rate and arrhythmias.

Holter ECG recordings obtained form 11 hyperthyroid patients (mean age 29.2 +/- 11.4 years) before and after surgical treatment were analyzed regarding heart rate (HR) and arrhythmias. The mean 24-hour HR significantly decreased form 104 +/- 8.8 bpm before to 76.8 +/- 8.9 bpm after the treatment (p less than 0.001), with the most pronounced reduction while asleep form 0:00 AM to 6:00 AM. HR variability also significantly decreased (p less than 0.02), but the circadian rhythm of HR did not change before and after the treatment. Serum T3 level correlated with the mean HR (r = 0.63, p less than 0.05) in hyperthyroid state. The percent changes in T3 (% delta T3) best correlated with the percent changes in the mean HR (% delta mean HR) (r = 0.84, p less than 0.01). The frequencies of supraventricular and ventricular premature contractions significantly decreased after the surgical treatment (p less than 0.01). Normal circadian pattern of hourly HR before the treatment highly suggests that normal adrenergic responsiveness is maintained in hyperthyroidism.

Adult↗

PubMed1991

Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli.

Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively. We already isolated and sequenced a cDNA clone encoding the type L isozyme. Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme. The message encodes a polypeptide of 838 amino acid residues. Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension. Except for these extra portions, the two isozyme sequences show an identity of 63%. The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b. Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C. The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C. Addition of pyridoxine to the culture medium was effective for the enzyme production.

Amino Acid Sequence↗

PubMed1991

Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals.

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.

Affinity Labels↗

PubMed1991

Expression in Escherichia coli of UDP-glucose pyrophosphorylase cDNA from potato tuber and functional assessment of the five lysyl residues located at the substrate-binding site.

The entire structural gene for potato tuber UDP-glucose pyrophosphorylase has been amplified from its cDNA by the polymerase chain reaction and inserted into the expression plasmid pTV118-N downstream from the lac promoter. Escherichia coli JM105 cells carrying thus constructed plasmid produced the enzyme to a level of about 5% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The recombinant enzyme purified to homogeneity in two column chromatographic steps was structurally and catalytically identical with the enzyme purified from potato tuber except for the absence of an N-terminal-blocking acetyl group. To examine functional roles of the five lysyl residues that had been identified by affinity labeling studies to be located at or near the active site of the enzyme [Kazuta, Y., Omura, Y., Tagaya, M., Nakano, K., & Fukui, T. (1991) Biochemistry (preceding paper in this issue)], they were replaced individually by glutamine via site-directed mutagenesis. The Lys-367----Gln mutant enzyme was almost completely inactive, and the Lys-263----Gln mutant enzyme had significantly decreased Vmax values with perturbed Km values for pyrophosphate and alpha-D-glucose 1-phosphate. Lys-329----Gln also exhibited increased Km values for these substrates but exhibited Vmax values similar to those of the wild-type enzyme. The two mutant enzymes Lys-409----Gln and Lys-410----Gln showed catalytic properties almost identical with those of the wild-type enzyme. Thus, among the five lysyl residues, Lys-367 is essential for catalytic activity of the enzyme and Lys-263 and Lys-329 may participate in binding of pyrophosphate and/or alpha-D-glucose 1-phosphate.

Amino Acid Sequence↗

PubMed1991

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells.

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.

Adenosine Diphosphate↗

PubMed1991

Role of leucine 66 in the asymmetric recognition of substrates in chicken muscle adenylate kinase.

Adenylate kinase has two distinct binding sites for nucleotide substrates, MgATP and AMP. To identify the location of the site that specifically interacts with the adenine ring of AMP, we have substituted Ala, Gly, Val, Gln, and Trp for Leu66 of the recombinant chicken muscle enzyme by site-directed mutagenesis. All the purified Leu66 mutant enzymes exhibited an essentially identical circular dichroism spectrum and had thermal stabilities similar to the wild-type enzyme. Steady state kinetic analysis showed that the Leu66 mutant enzymes have significantly decreased Vmax values and markedly large Km values only for AMP. These results show that the binding site for the adenine ring of AMP in adenylate kinase is presumably located close to Leu66, which is invariant in all the enzymes so far sequenced. Significant inhibition of activities of the mutant enzymes and quenching of the Trp66 fluorescence by substrates suggest that in some Leu66 mutant enzymes, MgATP also binds to the AMP-binding site. Thus, Leu66 of adenylate kinase might play a role in the asymmetric recognition of the adenine ring of AMP from that of MgATP. Furthermore, the hydrophobicity of the residue at position 66 appears to be important for the positive cooperativity of substrate binding.

Adenosine Monophosphate↗

PubMed1991

Catalytic site of F1-ATPase of Escherichia coli. Lys-155 and Lys-201 of the beta subunit are located near the gamma-phosphate group of ATP in the presence of Mg2+.

The catalytic site of Escherichia coli F1 was probed using a reactive ATP analogue, adenosine triphosphopyridoxal (AP3-PL). For complete loss of enzyme activity, about 1 mol of AP3-PL bound to 1 mol of F1 was estimated to be required in the presence or absence of Mg2+. About 70% of the label was bound to the alpha subunit and the rest to the beta subunit in the absence of Mg2+, and the alpha Lys-201 and beta Lys-155 residues, respectively, were the major target residues (Tagaya, M., Noumi, T., Nakano, K., Futai, M., and Fukui, T. (1988) FEBS Lett. 233, 347-351). Addition of Mg2+ decreased the AP3-PL concentration required for half-maximal inhibition, and predominant labeling of the beta subunit (beta Lys-155 and beta Lys-201) with the reagent. ATP and ADP were protective ligands in the presence and absence of Mg2+. The alpha subunit mutation (alpha Lys-201----Gln or alpha Lys-201 deletion) were active in oxidative phosphorylation. However, purified mutant F1s showed impaired low multi-site activity, although their uni-site catalyses were essentially normal. Thus alpha Lys-201 is not a catalytic residue, but may be important for catalytic cooperativity. Mutant F1s were inhibited less by AP3-PL in the absence of Mg2+, and consistent with this, modifications of their alpha subunits by AP3-PL were reduced. AP3-PL was more inhibitory to the mutant enzymes in the presence of Mg2+, and bound to the beta Lys-155 and beta Lys-201 residues of mutant F1 (alpha Lys-201----Gln). These results strongly suggest that alpha Lys-201, beta Lys-155, and beta Lys-201 are located close together near the gamma-phosphate group of ATP bound to the catalytic site, and that the two beta residues and the gamma-phosphate group become closer to each other in the presence of Mg2+.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.

Amino Acids↗

PubMed1991

Fluctuation of fetal rat hepatic histidine decarboxylase activity through the glucocorticoid-ACTH system.

Our studies suggest that the fluctuation of HDC activity in fetal liver in late gestation is regulated by the plasma glucocorticoid level through the pituitary-adrenal system. Taken together, these results support the conclusion that glucocorticoid promotes a rapid increase in HDC synthesis in fetal liver histamine-forming cells, as well as in mouse mastocytoma P-815 cells and rat glandular stomachs.

Adrenocorticotropic Hormone↗

PubMed1991

A bacteriological study of perforated duodenal ulcers.

Peritoneal fluid sampling and bacteriological examination were performed in 63 patients with perforated duodenal ulcers, and the results compared with those in 175 patients with other perforations. Bacterial culture was positive in 100 per cent of the patients whose perforations occurred in the colon, whereas it was positive in only 44.4 per cent of those with duodenal perforations, being negative in many cases when the interval from perforation to surgery was short. A mixed contamination with both aerobes and anaerobes was usually found in the cases of lower digestive tract perforation, and the isolates from duodenal perforations were uniquely aerobes in most cases. It is suggested that bacteria play a minor role in the pathogenesis of early stage duodenal perforation, which supports the technique of primary closure without indwelling drainage tubes during early stage operations following sufficient peritoneal lavage. Moreover, if the stomach is empty at the time of perforation and the peritonitis is localized, even conservative therapy seems possible, provided it is begun shortly after the perforation.

Adolescent↗

PubMed1991

The relationship between juxtapapillary duodenal diverticula and the presence of bacteria in the bile.

A total of 432 patients with gallstone disease were studied with respect to the existence of juxtapapillary duodenal diverticula and their relationship to the presence of bacteria in the bile. A total of 63 patients were found to have diverticula with an incidence of 14.6 per cent, being significantly higher in the elderly group aged 60 years or older (p less than 0.01), and no sex difference was noted. Among the patients with diverticula, positive bacterial cultures of bile were recognized at a significantly higher frequency, being found in 49 of the 63 patients (77.8 per cent; p less than 0.01), and the probability of bilirubinate stones was also higher, being found in 35 of 37 patients (94.6 per cent; p less than 0.01). The presence of a diverticulum bore significant relation to a higher positive bile bacterial culture (p less than 0.05), dilation of the common bile duct (p less than 0.05), and elevation of the bile duct pressure (p less than 0.05), even when the conditions were divided into cholecystolithiasis or choledocholithiasis. It was suggested that the presence of a diverticulum affected the flow in the bile duct by narrowing it from the outside and chronically stimulating the papilla, inducing biliary tract infection and/or the formation of gallstones. As the surgical procedures for juxtapapillary duodenal diverticula, including its indications, have not been established, long term follow up investigations seem necessary.

Adult↗

PubMed1991

Activation of phosphoinositide-specific phospholipase C delta from rat liver by polyamines and basic proteins.

Phospholipase C from rat liver with a molecular weight of 87,000 (PLC delta) is stimulated by polyamines, basic proteins, and basic polyamino acids. The activation occurs in both the presence and the absence of detergents. Half-maximum activation by spermine is observed at 0.15 mM, with optimum effects between 0.2 and 0.5 mM. Spermine inhibits above 0.5 mM. Half-maximum activation by spermidine and putrescine is observed at 0.9 and 6 mM, respectively, with optimum effects at 2 and 5 mM, respectively. These polyamines also inhibit at higher concentrations. Neomycin activates the enzyme with an optimum concentration of 10 microM, but maximum activation is less than with polyamines. Half-maximum activation by histone 2B occurs at 0.5 micrograms/ml (36 nM), with maximum stimulation at 1.5 micrograms/ml. Other histones, protamine, melittin, poly-L-ornithine, poly-L-lysine, poly-D-lysine, and poly-L-arginine, activate optimally at 3-10 micrograms/ml. Myelin basic protein and lysozyme activate optimally at 50-100 micrograms/ml. Typical activations are three- to eightfold, but under some conditions the enzyme shows little or no activity in the absence of basic activators. The basic activators lower the salt concentration required for maximal activity. In the case of the detergent-micelle assay, histone shifts the optimum NaCl concentration from 350 to 200 mM for PIP2, from 260 to 100 mM for PIP, and from 150 to 0 mM for PI. Histone potentiates the activation by Ca2+, but does not shift the optimum Ca2+ concentration. The optimum salt and Ca2+ concentrations are linked, such that a decrease in the concentration of one decreases the optimum concentration of the other. Activation by histone is diminished by MgCl2 in a concentration-dependent manner.

Animals↗

PubMed1991

Comparative affinity labeling with reactive UDP-glucose analogues: possible locations of five lysyl residues around the substrate bound to potato tuber UDP-glucose pyrophosphorylase.

By using two reactive analogues of UDP-Glc, uridine di- and triphosphopyridoxals, we have recently probed the substrate-binding site in potato tuber UDP-Glc pyrophosphorylase [EC 2.7.7.9]. In this work, pyridoxal diphospho-alpha-D-glucose was used for the same purpose. This compound is also a reactive UDP-Glc analogue but having its reactive group on the opposite side of the pyrophosphate linkage to those of the above two compounds. The enzyme was rapidly inactivated when incubated with the compound at very low concentrations followed by reduction with sodium borohydride. The inactivation was almost completely prevented by UDP-Glc and UTP. Complete inactivation correspond to the incorporation of 1.0 mol of the reagent per mol of enzyme monomer. The label was found to be distributed in five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-40. All of these results were similar to those obtained previously with the other compounds, suggesting the presence of a cluster of five lysyl residues at or near the substrate-binding site of this enzyme. However, the incorporations of labels into each lysyl residue differed depending on the compounds used. The substrate retarded the incorporations in different manners. Based on the combined results of the present and previous studies, a hypothetical model is presented for the possible locations of the five lysyl residues around the substrate bound to the enzyme. This model is consistent with the kinetic properties of mutant enzymes in which the five lysyl residues were individually replaced by glutamine via site-directed mutagenesis.

Affinity Labels↗

PubMed1991

[A case of Pasteurella multocida subsp. multocida complicated with diabetes mellitus].

We report a case of sepsis who died caused by Pasteurella multocida subsp. multocide sepsis. A 68-year-old male was admitted to Azusawa Hospital because of disturbance of consciousness. He had been suffering from diabetes mellitus combined with gangrene, but received no treatment. The patient died 24 h after hospitalization, and Pasteurella multocida subsp. multocida was isolated from his blood. Laboratory tests showed that CRP; 5+ WBC; 15,400/microliters, TP; 5.2 g/dl. Although Pasteurella multicida subsp. multocida seemed to cause mild infection in healthy subjects, it can cause severe systemic illnesses such as sepsis and meningitis in compromised hosts. It should be considered that the contact with pets will increase the incidence of systemic severe infection with this agents.

Aged↗

PubMed1991

Plasma immunoreactive endothelin-1 in experimental malignant hypertension.

We measured plasma concentrations of immunoreactive endothelin-1 (irET-1) in the prehypertensive and hypertensive phases in spontaneously hypertensive rats (SHR) and in malignant hypertension caused by deoxycorticosterone acetate (DOCA)-salt administration in SHR. We also measured concentrations of this peptide in another model of malignant hypertension, the two-kidney, one clip (2K1C) renovascular hypertensive rats chronically given caffeine. Plasma irET-1 concentrations in young (6-week-old) and mature (18-week-old) SHR did not differ from those of age-matched Wistar-Kyoto (WKY) rats. Four weeks of treatment with DOCA-salt increased blood pressure, blood urea nitrogen, serum creatinine, and plasma irET-1 in SHR but not in WKY rats. Eight weeks of DOCA-salt treatment further increased these values in SHR. Plasma irET-1 concentrations were not increased in the 2K1C rats. Six weeks of caffeine administration increased blood pressure, blood urea nitrogen, serum creatinine, plasma renin activity, and plasma irET-1 in the 2K1C rats but not in the sham-operated rats. High-performance liquid chromatographic profiles of plasma extracts pooled from these rats with malignant hypertension showed that a major component of irET-1 eluted in the position of synthetic ET-1 (1-21). Furthermore, acute hypertension induced by angiotensin II or phenylephrine did not affect the plasma irET-1 concentration in rats. The results suggested that the plasma ET-1 concentration is increased in rat models of malignant hypertension and that the high blood pressure itself is not the main factor involved in the increase of plasma ET-1.

Aging↗

PubMed1991

The inhibitory effect of tiamulin on high K(+)-induced contraction in guinea pig intestinal smooth muscle.

Tiamulin with an IC50 of 1.7 x 10(-6) M inhibited both the rapid and sustained contractions induced by hyperosmotically added 60 mM K+ (Hyper 60 K+) without changing the membrane potential in the intestinal muscle. Tiamulin inhibition (2 x 10(-6)-2 x 10(-5) M) of the Ca(2+)-induced contraction in depolarized muscle was competitively antagonized by raising external Ca2+. Tiamulin (2 x 10(-5) M) slightly affected the Hyper 60 K(+)-induced phasic contraction under hypoxia and the carbachol-induced phasic contraction. Moreover, tiamulin (2 x 10(-5) M) inhibited the Hyper 60 K(+)-induced contraction with decreasing [Ca2+]cyt level. Although the inhibitory effect of 10(-7)-10(-5) M monesin, an inhibitor of mitochondrial respiration, on the Hyper 60 K(+)-induced contraction was reduced under hypoxia, the effect of tiamulin (2 x 10(-7)-2 x 10(-4) M) was not modified. Tiamulin changed neither the intracellular Na+ and K+ content of the depolarized muscle nor the Ca(2+)-induced contraction in the chemically skinned preparations. These results suggest that the inhibitory action of tiamulin on the Hyper 60 K(+)-induced tonic contraction is possibly due to the competitive inhibition of Ca2+ entry through the voltage-dependent Ca2+ channel of the intestinal smooth muscle cell.

Animals↗

PubMed1991

[Relationship between referral and patient characteristics].

In order to evaluate the relationship between referral and patient characteristics, 594 new patients utilizing Saga Medical School general clinic were studied using a questionnaire and chart review. Results obtained are as follows: 1) Fifty-eight patients (10%) came for follow-up of mass screening tests, 174 patients (29%) were self-referral, the remaining 362 patients (61%) were under previous care by other physicians. Only 64 patients (11%) had a referral letter. 2) Those patients who were not advised to visit our clinic by non-medical personnel, patients who were seriously ill, elderly patients, residents in distant area, and those with medical problems tended to have a referral letter. 3) Of the 64 patients having referral letters, 15 (23%) took the initiative in asking their previous doctor to write a referral letter. Patients who were highly educated had a greater tendency to request referral letters. 4) The reasons cited by the patients without a referral letter included "Did not think of the necessity" (22%), "Wanted to confirm diagnosis" (22%), "Did not want to make the doctor lose face" (16%), "No improvement in symptoms" (14%) These results indicate that education of patients and the general public, and establishing good physician-patient communication and trust are necessary for increasing the use of referral letter.

Adult↗

PubMed1991

[Studies on epidemiology of nontuberculous mycobacteriosis--on the regional difference of the incidence of pulmonary diseases due to M. kansasii and M. avium complex in Tokyo area].

On 397 patients with pulmonary disease due to nontuberculous mycobacteria (NTM) [102 due to M. kansasii (MK) and 295 due to M. avium complex (MAC)] observed at National Tokyo Chest Hospital, 191 patients with pulmonary disease (59 due to MK and 132 due to MAC) observed at Fukujuji Hospital of Antituberculosis Association and 257 patients from whose sputum MK (36) or MAC (221) were isolated in Byotai-Seiri Clinical Laboratory, the distribution of these patients by domicile in Tokyo area was analysed. The percentage of patients with MK disease among the whole patients with MK disease and MAC disease (MK ratio) in each community area was investigated. MK ratio was 30.9% in the 23 wards and 16.4% in Tama section of Tokyo in the patients observed at National Tokyo Chest Hospital. It was 37.0% in the 23 wards and 32.1% in Tama section in the patients observed at Fukujuji Hospital and was 17.7% in the 23 wards and 8.1% in Tama section in the patients observed at Byotai-Seiri Clinical Laboratory. MK ratio in Tokyo was considerably higher in the 23 wards that were densely populated industrial and commercial areas than in Tama section, a comparatively sparsely populated suburb. MK ratio in patients of Ota Hospital located in the 23 wards was higher (36.4%) than that of Tachikawa-Sogo Hospital located in Tama area (10.0%). Regional differences in MK ratio were remarkable in Tokyo area. A high MK ratio appeared to correlate with a high incidence rate of tuberculosis. From the results mentioned above, it was suggested that M.K. disease may be transmitted from person to person.

Humans↗