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Biomedical subjects

T Horiuchi

Publications and source records attributed to T Horiuchi.

At least 361 records · Page 20Linked to original sources
PubMed1990

[A case of major dehiscence of a prosthetic aortic valve].

A case, 48-year-old man, was presented of dehiscence of an aortic Björk-Shiley prosthesis, which was diagnosed by pendular movement of prosthetic mount in cineangiographic finding. Emergent operation of Re-AVR was performed successfully. Valve dehiscence should be confirmed by cineangiographic examination, in case of progressive congestive heart failure combined with traumatic hemolytic anemia and high grade fever suggestive of prosthetic valve endocarditis.

Aortic Valve↗
PubMed1989

Purification of a DNA replication terminus (ter) site-binding protein in Escherichia coli and identification of the structural gene.

In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells. With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold. Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter protein revealed a specific binding to the ter sequences.

Amino Acid Sequence↗
PubMed1989

Mast cells are important in the development of hypersensitivity pneumonitis. A study with mast-cell-deficient mice.

We have previously reported that C57B1/6 mice develop lung lesions similar to human hypersensitivity pneumonitis (HP) by repeated transnasal administration of Thermoactinomyces vulgaris antigen. Since the HP-like lesions were induced via respiratory route and by the causative antigen in human HP (farmer's lung), it seems that this murine model is useful for investigating the cell-to-cell interactions in human HP. To clarify the involvement of mast cells (MC) in the development of HP, T. vulgaris (90 micrograms/day) was transnasally administered to MC-deficient WBB6F1-W/Wv mice (W/Wv) and their littermates (+/+) five times a wk for 3 wk. When the lungs were examined by scoring pathological findings and lung indexes, HP-like lesions were significantly less severe in W/Wv than in +/+, whose lesions were equivalent to those of C57B1/6. Bone-marrow-derived cultured MC from +/+ mice (98% purity) were obtained by in vitro culture mixed with WEHI-3B-derived conditioned medium which contained IL-3. When these MC were adoptively transferred to W/Wv mice (10(7) cells/mouse), the HP-like lesions in W/Wv mice were enhanced to be as severe as those in +/+. Importantly, significant numbers of MC were found in the lungs of MC-transferred W/Wv mice. These results suggest that MC play an important role in the development of the murine experimental HP.

Alveolitis, Extrinsic Allergic↗
PubMed1989

cDNA cloning and expression of human complement component C2.

A full-length cDNA clone for C component C2 was isolated from a human liver cDNA library in lambda gt11 by initially screening with an affinity-purified rabbit anti-C2 antibody and then using the isolated partial C2 cDNA as a probe for re-screening the library. The cDNA insert of clone lambda C2HL5-3 was sequenced in its entirety. It consisted of 2961 nucleotides including a 5' untranslated region of 388 nucleotides, followed by a 60 nucleotide region coding for a putative signal peptide, a 2196 nucleotide region coding for the 732 amino acids of the mature C2 polypeptide, and a 317 nucleotide long 3' untranslated region. Comparison of the nucleotide sequence to the previously reported C2 cDNA sequence showed two major differences. First, the 5' untranslated region of C2HL5-3 was 352 nucleotides longer and included four ATG followed by in-frame termination codons. Second, nucleotide residue 1987 was a C instead of a G, resulting in a change of amino acid residue 513 from Leu to Phe and in the appearance of an EcoRI site. The full-length C2 cDNA was cloned into the expression vector p91023(B). Transfection of the recombinant plasmid in COS cells resulted in the secretion of a protein with antigenicity and hemolytic activity similar to those of native C2. Western blot analysis indicated that secreted rC2 had the same apparent m.w. as C2 in human serum. Northern blot analysis of total RNA isolated from transfected COS cells showed two bands of C2 mRNA, both of which were longer than human liver C2 mRNA and represent transcripts generated by the vector-C2 construct.

Amino Acid Sequence↗
PubMed1989

Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli.

Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.

Autoradiography↗
PubMed1989

Inhibitory effect of free fatty acids on plasma protein binding of disopyramide in haemodialysis patients.

The plasma (or serum) protein binding of disopyramide (DSP) in five haemodialysis patients was studied using an ultrafiltration technique. There was an increase in the free fraction of DSP in the plasma on dialysis days in comparison to the levels on interdialysis days, which was associated with an elevation of the free fatty acid levels in the plasma together with the increase of the free fraction of DSP. The inhibitory effect of free fatty acids on DSP binding in an in vitro study was enhanced in proportion to their concentrations, and was shown to be due to competition at one binding site by experiments with oleic acid as a representative displacer. Certain endogenous organic acids, such as indoxyl sulphate, 2-hydroxyhippuric acid and hippuric acid, which are characteristically elevated in chronic renal failure, scarcely affected the protein binding of DSP. The findings indicate that free DSP should be monitored in patients with elevated plasma free fatty acid levels, such as those on haemodialysis therapy.

Adult↗
PubMed1989

Activation of protein kinase C induces differentiation in the human T-lymphoblastic cell line MOLT-3.

We attempted to determine whether or not activation of calcium phospholipid-dependent protein kinase C (PKC) is associated with the induction of differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the human T-lymphoblastic cell line MOLT-3. PKC activities were assayed in MOLT-3 and its five subclones resistant to TPA-induced cell differentiation. The cytosolic PKC activities of TPA-resistant subclones were 36-53% of that of the parental MOLT-3 cells. TPA treatment led to a rapid decrease in PKC activities in the cytosol, together with a concomitant increase in PKC activities in the particulate fraction, in both MOLT-3 and a TPA-resistant subclone. Thus, translocation of PKC from the cytosol to the membrane occurred following treatment with TPA, in both cell lines. However, the amount of PKC translocated from the cytosol to particulate fraction for 60 min in a TPA-resistant subclone was about 20% of that of the parental MOLT-3 cells. These findings suggest that the quantity of cytosolic PKC activity and the extent of translocation may relate to responses to TPA-induced cell differentiation in this T-cell line.

Cell Line↗
PubMed1989

A specific role of MutT protein: to prevent dG.dA mispairing in DNA replication.

Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants. With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity. The protein catalyzes hydrolysis of dGTP to dGMP. dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP. No other deoxynucleoside triphosphates were hydrolyzed. Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E. coli DNA polymerase III. When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated. The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair. Misincorporation of dAMP was not observed in reactions with the alpha subunit or core. The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture. Under these conditions, only a portion of dGTP present in the reaction mixture was degraded. It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine.

Adenine↗
PubMed1989

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.

Alanine↗
PubMed1989

Escherichia coli replication termination protein impedes the action of helicases.

Identification of the consensus sequence for termination of replication (ter) in Escherichia coli and the isolation of the ter-binding protein (TBP) allowed us to test their effects on replication forks initiated at the unique origin of the E. coli chromosome (oriC) in a purified enzyme system. Replication was severely impeded by ter in a unique orientation when purified TBP was supplied to bind it. The target for blockage within the replication complex can now be ascribed to the inability of dnaB helicase to separate the duplex strands when it encounters ter bound by TBP. Other helicases, such as rep and uvrD proteins, that translocate on DNA and displace strands in the direction opposite to that of dnaB protein are also blocked, but only when the TBP-bound ter is oriented in the other direction. From these results, we infer that the orientation of ter confers a particular polarity on the TBP seated on it, such that a helicase is blocked when it confronts TBP from one side, but can act, presumably by displacing TBP, when facing its other side. Thus, the intrinsic nature of the oriented TBP-ter complex is responsible for impeding the helicases, rather than any protein-protein interactions.

Base Sequence↗
PubMed1989

Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida.

Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin. The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al. (1986) J. Bacteriol. 166, 1089-1095). Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB). These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.

Amino Acid Sequence↗
PubMed1989

Suppression of dnaE nonsense mutations by pcbA1.

DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.

Blotting, Western↗
PubMed1989

Spontaneous regression in murine hypersensitivity pneumonitis: lack of immunological tolerance.

We have reported a murine model of hypersensitivity pneumonitis (HP) by transnasal administration of Thermoactinomyces vulgaris (Tv) antigen and shown a potential usefulness in the study of the immunopathogenesis of HP. Since we observed spontaneous regression (SR) of the lung lesions with continuous administration of Tv antigen in our model of HP, attempts were made to study if SR is caused by immunological tolerance. The titers of anti-Tv IgG antibody in serum and bronchoalveolar lavage fluid (BALF) did not show any decrement during the period of SR, at the 6th and 9th weeks after the first administration of the antigen. A delayed-type hypersensitivity reaction, i.e. footpad swelling with Tv antigen, did not change significantly during the SR period. Analysis of BALF cells disclosed that Lyt-2-positive cells, i.e. suppressor/cytotoxic T lymphocytes, did not increase to predominate Lyt-1-positive cells. The lymphocyte proliferation test with Tv antigen showed that the lung lymphocytes reacted to Tv antigen as well as concanavalin A during the period of SR in a similar magnitude as in the development period (3 weeks after the first treatment with the antigen). Treatment with cyclophosphamide before the period of SR resulted in no delay of SR. Finally, the adoptive cell transfer of spleen T cells obtained from mice during the SR period did not manipulate the development of granulomatous pneumonitis in the recipient mice. We concluded that the events causing SR in our murine model of HP may not involve the induction of immunological tolerance in humoral or cellular immunity.

Alveolitis, Extrinsic Allergic↗
PubMed1989

Alveolar type II cells, surfactant protein A (SP-A), and the phospholipid components of surfactant in acute silicosis in the rat.

Silica instillation causes a massive accumulation of surfactant phospholipids and the appearance of hypertrophic type II cells in the rat. We have examined the metabolic consequences of silica instillation with a special emphasis on surfactant protein A (SP-A), the major glycoprotein of surfactant. Fourteen days after instillation of 10 mg of silica, the amount of phospholipids in lavage increased 14-fold and the amount of SP-A increased 10-fold. The phospholipid composition of lavage material from silica-treated animals was altered by a reduction in the percentage of phosphatidylglycerol and an increase in phosphatidylinositol. The percentage of saturated phosphatidylcholine did not change. Type II cells isolated from rats treated with silica could be separated into cells of normal size and of increased size by centrifugal elutriation. There was an increase in phospholipid and SP-A content in the larger type II cells isolated from silica-treated rats relative to type II cells from control rats (by 45% and 70%, respectively, when expressed per micrograms protein; p less than 0.01). Activities of the phospholipid biosynthetic enzymes cholinephosphate cytidyltransferase, lysophosphatidylcholine acyltransferase, and phosphatidylglycerol phosphate synthetase increased (nmol/min/mg cell protein) in the hypertrophic type II cells relative to type II cells from control rats (by 40%, 112%, and 95%, respectively, p less than 0.05). Incorporation of (1-14C)-acetate was also increased in hypertrophic type II cells relative to type II cells from control rats (by 43%, p less than 0.05). The only difference in the distribution of acetate incorporated into individual lipids was a slight increase in the percentage incorporated into phosphatidylinositol. Although the increase in phosphatidylcholine found in the lavage could be due to increased synthesis as reflected by the changes in enzymatic activity and rate of acetate incorporation, the reduction in phosphatidylglycerol in lavage and in type II cells could not be explained simply by a reduction in synthesis on the basis of available data. There was a marked increase in SP-A content in lavage and in type II cells isolated from silica-treated rats without a significant change in the relative abundance of SP-A mRNA. This dissociation of SP-A mRNA abundance and apoprotein content suggests that factors other than transcription may be important for the observed accumulation of SP-A in silica-treated rats. We conclude that the phospholipidosis and proteinosis that occur subsequent to silica instillation in the rat lung are not solely attributable to increase in the rates of synthesis of these components.

Acute Disease↗