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Biomedical subjects

T Horiuchi

Publications and source records attributed to T Horiuchi.

At least 19 recordsLinked to original sources
PubMed1992

Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme.

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].

Amino Acid Sequence↗
PubMed1992

Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme.

Glu35 in chicken lysozyme has an abnormally high pKa (6.1) partly due to the hydrophobic environment provided by Trp108. The relationship between protein stability and abnormal pKa was investigated in detail by using mutant lysozymes in which Glu35 was replaced by undissociable residues and an oppositely ionizable residue. It was found that lysozyme was stabilized at alkaline pH range by the replacement of Glu35 with an undissociable residue, Gln (E35Q lysozyme) or Al (E35A lysozyme). On the other hand, when Glu35 was replaced by His (E35H lysozyme), which could have an opposite charge to Glu by ionization, the introduced His35 was found to have an abnormally low pKa (3.6), leading to the destabilization of lysozyme at acidic pH. These observations are completely consistent with the situation that the environment around Glu35 is highly hydrophobic and therefore the placement of either a positive or negative charge in such an environment leads to destabilization of lysozyme. These observations also indicate that the replacement of an acidic residue having abnormally high pKa or a basic residue having abnormally low pKa by an undissociable residue is a very efficient and general method for stabilization of a protein.

Animals↗
PubMed1992

Intestinal type alkaline phosphatase hyperphosphatasemia associated with liver cirrhosis.

Hyperphosphatasemia due to increased intestinal type serum alkaline phosphatase was noted in a 48-year-old male who had asymptomatic liver cirrhosis. The alkaline phosphatase activity in the serum was 828 U/l (our reference range in adults: 57-194 U/l), 94% of which was of the intestinal type as measured by an immunoprecipitation method. The intestinal component of alkaline phosphatase was separated into two major and some minor components using electrophoresis and isoelectrofocusing. One of the major components had similar mobility to that of a standard intestinal enzyme purified from adult intestine. The components were heat-labile and neuraminidase-resistant. Serial lectin affinity chromatography, however, indicated that sugar chain compositions of the alkaline phosphatase were different from those of the standard tissue intestinal enzyme. These results and further enzymological studies suggest that the patient's serum alkaline phosphatase basically consisted of several intestine-like isoforms.

Alkaline Phosphatase↗
PubMed1992

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins.

The letA (ccdA) and letD (ccdB) genes of F plasmid contribute to stable maintenance of the plasmid in Escherichia coli cells; a product of the latter has a lethal effect on the host cell and that of the former neutralizes functions of the letD. In cells that overproduce the LetD (CcdB) protein, the plasmid DNA is extensively relaxed. Correspondingly, DNA supercoiling activity in a cell-free extract of the overproducing strain decreases to a level of less than 1% of that seen in normal cells. However, the extract does not inhibit DNA gyrase reconstituted from purified subunits, thereby indicating that the intrinsic DNA gyrase is inactivated in the overproducing strain. Upon addition of purified LetA (CcdA) protein to the extract of LetD overproducing cells, the DNA supercoiling activity was fully restored. Using this rejuvenation as an assay, we purified the "inactivated gyrase" and obtained evidence that the LetD protein formed an isolable complex with the A subunit of DNA gyrase. Thus, the LetD and the LetA proteins constitute an opposing pair in modulating the DNA supercoiling activity of gyrase, probably by direct interaction.

Bacterial Proteins↗
PubMed1992

Sequence and expression of rat ICAM-1.

We have isolated cDNA clones-coding for rat intercellular adhesion molecule-1 (RICAM-1) from a cDNA library constructed from rat Ax cells stimulated with IL-1 beta using the mouse ICAM-1 cDNA as a hybridization probe. The RICAM-1 sequence shows 79.1% homology with mouse ICAM-1 and 55.6% homology with human ICAM-1 at the nucleic acid level. In order to examine the expression of RICAM-1 on Chinese hamster ovary (CHO) cells, we constructed the vector, pSV-RICAM1-neo, containing the SV40 promoter. Flowcytometric analysis showed that CHO-K1 cells transfected with pSV-RICAM1-neo expressed high amounts of RICAM-1 on their surfaces.

Amino Acid Sequence↗
PubMed1992

Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit A suppress letD (ccdB) product growth inhibition.

The letA (ccdA) and letD (ccdB) genes, located just outside the sequence essential for replication of the F plasmid, apparently contribute to stable maintenance of the plasmid. The letD gene product acts to inhibit partitioning of chromosomal DNA and cell division of the host bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. To identify the target of the letD gene product, temperature-sensitive growth-defective mutants were screened from bacterial mutants that had escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Of nine mutants analysed, three mutants were shown, by phage P1-mediated transduction and complementation analysis, to have mutations in the gyrA gene and the other six in the groE genes. The nucleotide sequence revealed that one of the gyrA mutants has a base change from G to A at position 641 (resulting in an amino acid change from Gly to Glu at position 214) of the gyrA gene. The mutant GyrA proteins produced by these gyrA(ts) mutants were trans-dominant over wild-type GyrA protein for letD tolerance. The wild-type GyrA protein, produced in excess amounts by means of a multicopy plasmid, overcame growth inhibition of the letD gene product. These observations strongly suggest that the A subunit of DNA gyrase is the target of the LetD protein.

Amino Acid Sequence↗
PubMed1992

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions.

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met1), Ad(Met-1), Ad(Met-12), and Ad(Met6). The shoulder numbers represent the site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met-1), were purified from the cell lysate and the ratios of A414-to-A276 of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450SCC reduction catalyzed by Ad(Met6) was about 60% of that by Ad(Met1), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450SCC would need to be considered.

Adrenodoxin↗
PubMed1992

Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.

Antigens, Polyomavirus Transforming↗
PubMed1992

Design and synthesis of an alpha-helical peptide containing periodic proline residues.

A thirty-residue peptide (PERI COIL-1) has been designed with a new type of alpha-helical structure, which is capable of folding into an amphiphilic helix bending at 4 periodic prolines in the sequence. Two such helices should form a dimer by supercoiling about one another in an antiparallel direction in the design. With this arrangement, close packing between them is maintained through the hydrophobic interaction pattern called 'leucine zipper'. PERI COIL-1 has been obtained by solid-phase peptide synthesis, and characterized by circular dichroic spectroscopy, sedimentation equilibrium experiments and NMR. The result of the analyses shows that it preferentially forms a helical tetramer in aqueous solution.

Amino Acid Sequence↗
PubMed1992

Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae.

The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster. These are located at the 3' end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I. We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis. The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome. To define the site further, smaller fragments were subcloned into the YEp-type plasmid. A small 109 bp region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription. It overlaps an essential element of the recombinational hot spot HOT1.

Base Sequence↗
PubMed1992

Anticarcinogenic effects of (-)-epigallocatechin gallate.

BACKGROUND: Our research objective is to develop nontoxic cancer chemopreventive agents and to apply these agents in treating humans. We are identifying agents that inhibit the process of tumor promotion in two-stage carcinogenesis experiments on mouse skin. METHODS: We review (a) the inhibitory effect of penta-O-galloyl-beta-D-glucose (5GG) on tumor promotion by teleocidin, one of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters (5GG is structurally similar to (-)-epigallocatechin gallate (EGCG) and is isolated from hydrolyzed tannic acid); (b) the inhibitory effects of EGCG, the main constituent of Japanese green tea, on tumor promotion with two tumor promoters, teleocidin and okadaic acid, a non-TPA-type tumor promoter; (c) the mechanisms of action of EGCG, a single application of which reduced the specific binding of [3H]TPA and [3H]okadaic acid to a particulate fraction of mouse skin; and (d) the anticarcinogenic effects of EGCG on duodenal carcinogenesis induced by N-ethyl-N'-nitro-N-nitrosoguanidine in male C57BL/6 mice. EGCG is a nontoxic compound. CONCLUSION: We believe that the main constituent of Japanese green tea, EGCG, is a practical cancer chemopreventive agent available in everyday life.

Animals↗
PubMed1992

Clinical application of an active electrode using an operational amplifier.

An active electrode (d10 mm, t6 mm) is presented, that functions as an impedance transformer (an input impedance > 10 G omega, an output impedance < 1 omega) by means of which we can derive surface EMG without any skin preparation and paste. This electrode was compared with a conventional one, and it was ascertained that the electrode could be replaced with the conventional one, and, moreover, it was preferable because it required less preparation time, and was less affected by environmental noise.

Amplifiers, Electronic↗
PubMed1992

Biological aspects of pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) in Sprague-Dawley rats. Immunohistochemical and ultrastructural studies.

Rat pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) were studied by the indirect peroxidase-labeled antibody method, together with ultrastructure and serum hormone measurement. Immunohistochemically, TZ-CT-induced pituitary tumors showed staining for only rLH alpha subunit, and were negative for other peptide hormones including GH, PRL, alpha MSH and ACTH, and the beta subunit of glycoprotein hormones. Electron microscopic examination showed that the majority of tumor cells possessed numerous small secretory granules, 100 to 200 nm in diameter. The serum PRL concentrations of rats with TZ-CT-induced pituitary tumors were markedly elevated, but not beyond 130 ng/ml. From our data, TZ-CT-induced pituitary tumors are considered to be endocrinologically inactive and to produce alpha subunit. Furthermore, these tumors are thought to be potentially useful models of alpha subunit-producing pituitary tumors in humans. This is the first report to document the tumorigenesis of alpha subunit-producing pituitary tumors in rats after long-term treatment with calcitonin.

Animals↗
PubMed1992

Idiopathic pulmonary fibrosis and high prevalence of serum antibodies to hepatitis C virus.

The prevalence of serum antibodies to hepatitis C virus (HCV) was assessed by an enzyme-linked immunosorbent assay (ELISA) in 66 patients (46 male and 20 female; mean age +/- SEM, 61.5 +/- 10.1 yr) with idiopathic pulmonary fibrosis (IPF). Nineteen (28.8%) were positive for this test. The frequency of HCV positiveness was significantly higher in the patients with IPF than in the 9,464 control subjects, whose ages were comparable with those of the patients (3.66%, p less than 0.05). Importantly, 12 of the 19 patients with IPF and positive ELISA results (63.2%) had positive results on the Chiron recombinant immunoblotting assay (RIBA), which is known to be more specific for HCV. We judged the 12 perceptible reactions as eight reactive and four indeterminate. When we examined liver function retrospectively, only two of eight patients who tested positive for the HCV had liver dysfunction, suggesting that anti-HCV positivity in IPF was not observed as a result of liver disease. These results lead us to speculate that HCV infection may play an important role in the pathogenesis of IPF, or that the sera of patients with IPF may contain some antibody against an unknown epitope and cross-react with the anti-HCV assay.

Aged↗
PubMed1992

Hypersensitivity pneumonitis in athymic nude mice. Additional evidence of T cell dependency.

We previously demonstrated that C57 Black/6 mice develop lung lesions similar to human hypersensitivity pneumonitis (HP) by repeated transnasal administration of Thermoactinomyces vulgaris (Tv) antigen. To elucidate the role of T cells in the development of this disease, Tv antigen (90 micrograms/day) was transnasally administered to athymic nude C57 Black/nu/nu (nu) mice and their littermates (+/nu) three times a week for 3 wk. The nude mice developed minimal lung lesions, whereas their thymus-intact littermates (+/nu) showed changes equivalent to those in C57 Black/6. Changes in local inflammatory cell responses were evaluated by bronchoalveolar lavage, and increases in the numbers of lymphocytes and macrophages were significantly less severe in the nude mice than in the +/nu mice. Interestingly, the increase in polymorphonuclear leukocytes 6 h after the last antigen inoculation was equivalently seen in both groups. When spleen-derived T cells (more than 95% Thy-1.2+) from the sensitized +/nu mice were adoptively transferred to nude mice, the HP-like lesions in the recipients were found after Tv antigen challenge. These results suggest that Thy-1.2+ T cell-mediated immunity was necessary for the development of HP in this murine model.

Alveolitis, Extrinsic Allergic↗
PubMed1992

Machine perfusion of isolated kidney at 37 degrees C using pyridoxalated hemoglobin-polyoxyethylene (PHP) solution, UW solution and its combination.

To preserve isolated kidney normothermically, PHP containing UW components were evaluated as perfusates. Kidneys were flushed out by Lactate Ringer solution immediately after isolation from mongrel dogs, and then connected to the perfusion circuit which consists of a preservation box, a reservoir of perfusate, a membrane oxygenator and a drive unit. PHP containing 140 mEq/l of Na+ and 4 mEq/l of K+ (PHP(E)), UW solution (UW) and UW components added PHP(E) were prepared and adjusted at pH 7.4 prior to use. Temperature and perfusion pressure were controlled at 37 degrees C and 100 mmHg, respectively. During 12 hour perfusion, remarkable changes in pH were seen in UW group and PHP group while higher oxygen consumption was noted in PHP(E)+UW group than that in PHP(E) group. The histological findings showed moderate damages of tubular epithelial cells and maintaining normal glomerular structure in PHP(E)+UW while severe damage of both tubulus in UW group were seen. There was no edematous degeneration in both UW and PHP(E)+UW groups, however, it was seen in PHP(E) alone. It was suggested that components of UW solution have positive effect on normothermic machine perfusion with PHP(E) solution.

Adenosine↗