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Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 343 records · Page 19Linked to original sources
PubMed1989

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal.

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.

Adenine Nucleotides↗
PubMed1989

Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF).

cDNA clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) were isolated and sequenced. The amino acid sequence deduced from the nucleotide sequence reveals the primary structure of the MCAF precursor to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. The amino acid sequence of MCAF showed 25-55% homology with other members of an inducible cytokine family, including macrophage inflammatory protein and some putative polypeptide mediators known as JE, LD78, RANTES and TCA-3. This suggests that MCAF is a member of family of factors involved in immune and inflammatory responses.

Amino Acid Sequence↗
PubMed1989

Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase.

Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.

Adenylate Kinase↗
PubMed1989

Illness behavior of housewives in a rural area in Japan: a health diary study.

A health diary study was conducted to examine the incidence and nature of health problems and illness behavior among rural residents in Japan. Attention was paid in particular to the utilization of folk medicine or alternative practitioners in the context of illness behavior. One hundred and sixty-one health problems were recorded over a 4-week diary period by 28 housewives aged 35-64 years. Headache, tiredness and gastrointestinal problems were among the most common problems. Emotional/psychological problems, the most frequently recorded problems in the health diary studies conducted in the United States or England, were recorded by only 3 participants. Only 6 problems (3.7 percent) resulted in consulting a doctor. Three women utilized an acupuncturist, shinkyushi, during the diary period. Self-care, such as resting by lying down, using home remedies and self-medication including household drugs, Toyama kusuri and folk medicine, was practiced for 101 problems (62.7 percent). Folk medicine or alternative practitioners played important roles in the health seeking process. The health diary method was shown to be suitable not only to Western communities but also in a rural Japanese context.

Adult↗
PubMed1989

Postoperative infection prophylaxis for upper gastrointestinal tract surgery--a prospective and comparative randomized study of cefoxitin and ceftizoxime.

A prospective and randomized clinical study was conducted in order to compare cefoxitin (CFX) and ceftizoxime (CZX) as prophylactic antibiotics. Two hundred and three consecutive cases of elective upper gastrointestinal tract surgery, performed at our institute between January, 1983, and March, 1986, were entered in the trial. The patients were assigned randomly, before surgery, to the CFX or to the CZX group. Two grams of the assigned antibiotic was first administered during surgery and then continued at a dose of 1 gram, every 8 hrs for a total of 4 days. One patient was withdrawn from the study due to an allergic reaction. Both groups were comparable in sex, age, underlying disease, diagnosis, operation, and preoperative laboratory data. There were 18 infections related to the operation in the CFX group, while there were only 3 in the CZX group (p less than 0.001). The number of unrelated infections in each group was 6 and 6 respectively (NS). No special differences were found between the two groups regarding the kinds of microorganisms isolated, and no significant differences were seen in the adverse effects of either antibiotic. Our study demonstrated that ceftizoxime was more efficient than cefoxitin in preventing postoperative infection, following upper gastrointestinal tract surgery.

Adult↗
PubMed1989

Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure.

We previously purified an auxin-binding protein (ABP) from the microsomal fraction of maize shoots (Zea mays L. cv. Golden Cross Bantam). In the present study cDNA clones derived from mRNAs encoding the ABP were isolated and sequenced. The nucleotide sequence of the 822-base-pair cDNA includes a 603-base-pair open reading frame. RNA blot hybridization analysis indicated a single mRNA species of approximately 1.0 kilobase. The predicted precursor of ABP is composed of 201 amino acid residues and has a molecular weight of 21,976. The NH2-terminal sequence of 38 residues is hydrophobic and may be a signal peptide for translocation of the ABP across the membrane of the endoplasmic reticulum. The mature ABP, composed of 163 residues with a molecular weight of 18,352, contains a potential N-glycosylation site (Asn-Thr-Thr), and the COOH-terminal tetrapeptide (Lys-Asp-Glu-Leu) may be a signal for retention of the ABP in the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1989

Site-directed mutagenesis of Gly-15 and Gly-20 in the glycine-rich region of adenylate kinase.

Glycine-15 or Glycine-20 in the glycine-rich region of chicken adenylate kinase was replaced by Ala via site-directed mutagenesis. The two mutant enzymes showed lower enzymatic activities under the standard assay conditions. Kinetic analyses of the mutant enzymes revealed that they have markedly lower affinities for AMP and ATP, the Vmax values being comparable to that of the wild-type enzyme. These properties are similar to those of the (Pro-17----Leu, Gly or Val) enzymes (Reinstein, J., Brune, M., & Wittinghofer, A. (1988) Biochemistry 27, 4712-4720; Tagaya, M., Yagami, T., Noumi, T., Futai, M., Kishi, F., Nakazawa, A., & Fukui, T. (1989) J. Biol. Chem. in press), providing evidence that the glycine-rich region in adenylate kinase is important for the binding of both substrate nucleotides. The substrate specificity and the susceptibilities as to thermal denaturation and proteolysis were also affected by the mutations.

Adenosine Triphosphatases↗
PubMed1989

Production and characterization of recombinant human neutrophil chemotactic factor.

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.

Amino Acid Sequence↗
PubMed1989

UDP-glucose pyrophosphorylase from potato tuber: purification and characterization.

UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30%. The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid. Product inhibition studies revealed the sequential binding of UDP-glucose and MgPPi and the sequential release of glucose-1-phosphate and MgUTP, in this order. Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPPi and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor. The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus. For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced. The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities.

Amino Acid Sequence↗
PubMed1989

Molecular cloning of cDNA encoding potato amyloplast alpha-glucan phosphorylase and the structure of its transit peptide.

The type L isozyme of potato tuber alpha-glucan phosphorylase [EC 2.4.1.1], a dimer of 104-kDa subunits, is compartmentalized in the amyloplast. We have cloned a nearly full-length cDNA encoding this isozyme from a cDNA library of immature potato tuber. The sequence was supplemented by a partial genomic clone. The transcription initiation site was identified by a primer extension experiment to be 43 bases upstream from the translation initiation ATG codon. The message encodes a polypeptide of 966 amino acid residues, of which 50 residues constitute an N-terminal extended peptide and 916 residues make up the mature protein. In the mature protein region, the nucleotide sequence is consistent with the chemically determined amino acid sequence (Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236). The N-terminal extension bears characteristic features of the transit peptides of nuclear-encoded chloroplastic proteins, and is therefore regarded as a transit peptide for the amyloplast. This peptide is rich in basic amino acids (5 arginines, 3 lysines, and 5 histidines) and hydroxylic amino acids (7 serines and 5 threonines), but lacks acidic amino acids. It is therefore classified as one of the most basic transit peptides so far reported.

Base Sequence↗
PubMed1989

Ca2(+)-dependent conformational change of the ATP-binding site of Ca2(+)-transporting ATPase of sarcoplasmic reticulum as revealed by an alteration of the target-site specificity of adenosine triphosphopyridoxal.

Adenosinetriphosphopyridoxal (AP3PL) specifically modifies Lys684 of Ca2(+)-ATPase of sarcoplasmic reticulum (SR-ATPase) in the presence of Ca2+, leading to its inactivation (Yamamoto, H. et al. (1988) J. Biochem. 103, 452-457). We have now investigated the effects of AP3PL on SR-ATPase in the absence of Ca2+. Similarly to its action in the presence of Ca2+, AP3PL inhibited the Ca2(+)-transporting activity in a dose-dependent manner in the absence of Ca2+ as well. ATP and ADP protected SR-ATPase against inactivation by this reagent. One mole of AP3PL was bound per mol of SR-ATPase with concomitant loss of the Ca2(+)-transporting activity. Binding of AP3PL to SR-ATPase was prevented by ATP. AP3PL-labeled SR membranes were digested with thermolysin and labeled thermolytic peptides were purified through C18 reversed-phase HPLC. Two major AP3PL-labeled peptides were obtained in approximately 1:1 ratio; one was an octapeptide corresponding to 679-ValGluProSerHisLys*SerLys-686, and the other, a nonapeptide corresponding to 487-PheSerArgAspSerLys*ArgMetSer-495 (Lys* indicates a labeled Lys residue) of SR-ATPase. Lys684 in the former turned out to be the same as the highly specific target of AP3PL in the presence of Ca2+ which was identified previously. The target site specificity of AP3PL thus changed significantly but not entirely on binding of Ca2+ to SR-ATPase. This indicates that the spatial arrangement around the gamma-phosphoryl group of the bound ATP is affected by Ca2+ ions bound at the transport site. It is also likely that Lys492 and Lys684 are situated close together in the ATP binding site of SR-ATPase.

Adenosine Triphosphate↗
PubMed1989

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.

Alcaligenes↗
PubMed1989

[Perioperative management of cesarean section in a patient pregnant with twins complicated by hyperthyroidism].

A 30-year-old twin pregnant woman complicated with hyperthyroidism was scheduled to receive anesthesia for cesarean section. During the early months of pregnancy, her thyroid status was stimulated, being positive for TSH-binding inhibitor immunoglobulins (TBII). Because maternal positiveness of TBII correlates with neonatal hyperthyroidism, she was treated with antithyroid drugs (methimazole). Her thyroid status was suppressed gradually and her TBII level became negative. On the 38th week of pregnancy, she underwent cesarean section by lumbar spinal anesthesia and twin girls were delivered with Apgar scores of 9. The thyroid statuses of the twins were measured, showing that their thyroid functions to be almost equal, although they were dizygotic twins. T3 and free-T3 were below the normal limits for adults, but they did not require any therapy. Maternal level of TBII is considered to be a useful index for management of patients with hyperthyroidism during the perianesthetic period for cesarean section. In this type of patients the thyroid status of both the mother and fetus should be carefully followed.

Adult↗
PubMed1989

[Spinal cord compression by primary epidural non-Hodgkin lymphoma].

A 63-year-old man was admitted because of sudden paraparesis. Myelography revealed a complete block at the level of T-3. The spinal epidural tumor was histologically diagnosed as malignant lymphoma, diffuse large cell type and immunohistochemically B-cell type. A thorough work-up for metastasis including MRI was negative. He was postoperatively treated with combination chemotherapies (VEPA), followed by irradiation to the region. The residual tumor disappeared on chest-CT and the patient's neurological status rapidly improved. He is still in remission for 18 months and has full performance status. This case is a very rare lymphoma originated from spinal-epidural space.

B-Lymphocytes↗
PubMed1989

[A case of primary pulmonary cryptococcosis showing spontaneous recovery].

A 37-year-old woman was admitted with fever and cough. The chest roentgenogram revealed a solitary large round mass in the right lower lung field. Diagnosis was made by transbronchial lung biopsy. There was no evidence of abnormal cell-mediated immunity. The patient made an uneventful recovery and the chest X-ray showed progressive clearing without specific therapy. After 6 months, the mass lesion disappeared. This case suggests that specific therapy may not be necessary for all cases of primary pulmonary cryptococcosis.

Adult↗
PubMed1988

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate.

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.

Animals↗