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Biomedical subjects

T Horiuchi

Publications and source records attributed to T Horiuchi.

At least 307 records · Page 17Linked to original sources
PubMed1993

Effects of microbial products on glucose consumption and morphology of macrophages.

We studied the effects of microbial products on glucose consumption and morphology of macrophages which were elicited with thioglycollate medium. Macromolecules such as lipopolysaccharide (LPS), tumor promoters, and respiratory inhibitors increased macrophage glucose consumption without inducing evident morphological changes. The assay system was used to screen for active substances in culture broth extracts from actinomycetes. Among them, aureothin increased glucose consumption of macrophages and inhibited respiration of a rat mitochondrial fraction. Concanamycin A induced morphological changes of macrophages into needle-like shapes but not of cloned cells including the macrophage-like cells J774.1. This compound changed fibrosarcoma L929 cells into round shapes without affecting the shape of a nontransformed fibroblast, BALB/3T3 cells. Antimycin and concanamycin A increased tumor-killing activity of macrophages when added during the effector phase. These results suggest that this assay system is simple and sufficiently reproducible and thus usable for screening for modulators of macrophage function among natural products.

3T3 Cells↗
PubMed1993

[Clinical evaluation of age-related changes of bone mineral content of cortical and trabecular bones by dual energy QCT].

The potential capability of a new dual energy (DE) quantitative computed tomography (QCT) method, 4-equation 4-unknown method (DEQCT4E-4U), was evaluated for estimation of bone mineral density (BMD) as well as bone mineral content/cm width (BMC) in trabecular bone of lumbar vertebrae. Cortical BMD and BMC were also estimated by the single energy QCT method (SEQCT 80 kVp) and the age-related change of cortical BMD and the cortical BMC/trabecular BMC ratio was also studied. The result indicated that the estimation of the BMD combining analysis of BMC in trabecular bone obtained by the present method provides further knowledge concerning vertebral trabecular bone mineral status. The profiles of the age-related changes of the cortical BMD and cortical BMC/trabecular BMC ratio in females were markedly different from those in males. These results indicated that our method combining analysis of BMC (BMD) of cortical bone and trabecular bone was also found to provide valuable information to evaluate the vertebral bone mineral status.

Absorptiometry, Photon↗
PubMed1993

Thigh pain in cementless total hip replacement.

The incidence of thigh pain was investigated in 51 joints implanted with model Y or YII cementless total hip system. The 51 patients were followed for one or more years after surgery. Twenty-six out of 51 implanted with the model Y, and the other 25 with the model YII. Thigh pain appeared in 11 out of 26 patients (43%) replaced by model Y and in only 2 out of 25 (8%) replaced by model Y II. The difference in the incidence of thigh pain was attributed to the difference in the extent of the press fit of the stem, stress distribution and postoperative activity. To prevent thigh pain, a favorable press fit must be achieved during surgery, and the excessive weight-bearing must be restricted in postoperative course. In our improved stem design, stress is distributed uniformly on the interface between the bone and the stem resulting in reduction of thigh pain.

Adult↗
PubMed1993

[A graphic presentation of the anesthesiologist's action pattern].

The action patterns of anesthesiologists are different among individuals. We have developed a graphic presentation system for anesthesiologists' action patterns using computer graphics. The resulting representation is called "thought map". Data were obtained from 6 anesthesiologists (4 certified instructors and 2 new doctors) with a time interval of 2 years, regarding the use of 3 drugs (enflurane, sevoflurane and isoflurane). The "maps" were compared among individuals, dates and drugs. The following results were obtained: 1) significant differences existed among individuals, 2) during the 2-year time interval, 2 new drugs were introduced and the practice pattern alterations were seen in the maps, 3) for the 2 new anesthesiologists, the maps show the process of professional knowledge acquisition. We propose an index ("immovable rate"), denoting the proportion of the map where concentration of the drug didn't change. This index is to be used for evaluating human thinking.

Anesthesia, Inhalation↗
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↗