Biomedical subjects

Y Natori

Publications and source records attributed to Y Natori.

At least 91 records · Page 5Linked to original sources

PubMed1993

Production of dihydrofolate reductase by an improved continuous flow cell-free translation system using wheat germ extract.

We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase mRNA as the translated message. Continuous buffer flow and separation of the product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as the reaction vessel. The system worked for 19 hours and produced 1.52 nmol (27.4 micrograms) of enzymatically active dihydrofolate reductase.

Base Sequence↗

PubMed1993

The biosynthesis of a cytotoxic protein, alpha-sarcin, in a mold of Aspergillus giganteus. II. Maturation of precursor form of alpha-sarcin in vivo.

Since Aspergillus giganteus ribosomes are sensitive to their own product, alpha-sarcin, we studied how resistance of protein synthesizing machinery is conferred in the cells. Immunoblotting analysis of a cell-extract prepared from mycelia of alpha-sarcin-producing phase showed two anti-alpha-sarcin reactive proteins which have molecular weights of 18.5 KDa and 16.9 KDa. The former and the latter were identified as the precursor and the mature forms of alpha-sarcin, respectively. Electronmicroscopic observation examining the location in situ by labeling with protein A-gold suggested that alpha-sarcin-related proteins are exclusively localized in large and small vacuoles. The small vacuoles, distributed near cell wall, appear to be involved in maturation and in the secretion of the protein. The present results suggested that alpha-sarcin is synthesized in an inactive precursor form and segregated in the membrane compartment through the synthesis and the maturation of alpha-sarcin in the cells.

Aspergillus↗

PubMed1992

Phenylmethylsulfonyl fluoride stimulates proteolysis of nuclear proteins from chick liver.

The degradation of H1 histone and high mobility group (HMG) nonhistone proteins was stimulated when the homogenate from chick liver was incubated in the presence of phenylmethylsulfonyl fluoride (PMSF). Two proteinase inhibitors, elastatinal and chymostatin, significantly inhibited the PMSF-stimulated degradation of H1 histone and HMG proteins. On the contrary, other proteinase inhibitors like leupeptin, pepstatin, trypsin inhibitor, antipain, o-phenanthroline and EDTA had no effect on the degradation of the nuclear proteins. These results warn the researcher to be cautious while using PMSF for preparation of nuclear proteins such as H1 histone and HMG proteins.

Animals↗

PubMed1992

Molecular cloning and characterization of genes encoding rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and pancreatic secretory trypsin inhibitor (PSTI).

The genes encoding a rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and its structurally related peptide, rat pancreatic secretory trypsin inhibitor (PSTI), have been isolated and sequenced. The two genes share extremely high sequence similarity in the 5' flanking regions, suggesting that these regions may be responsible for the characteristic coordinate expression of the two peptides.

Amino Acid Sequence↗

PubMed1992

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.

Amino Acid Sequence↗

PubMed1992

An efficient expression system for a variant form of the cytotoxic protein alpha-sarcin in Escherichia coli.

An efficient Escherichia coli system for the production of a variant form of the cytotoxic protein alpha-sarcin (delta Ala 1) has been constructed. cDNA encoded alpha-sarcin lacking N-terminal alanine was ligated with the bla signal peptide sequence (the signal sequence of E. coli beta-lactamase localizes the protein in the periplasm) and was inserted into an inducible bacterial expression vector pKTN2-2. When the plasmid introduced into E. coli was expressed in the presence of IPTG, the recombinant product accumulated in the periplasmic space. The product was purified by Affi-Gel Blue followed by Bio-Rex 70 column chromatography. Recombinant alpha-sarcin (delta Ala 1) displayed the properties similar to those of authentic alpha-sarcin isolated from Aspergillus giganteus with respect to its molecular mass and enzymatic activity in ribosomal inactivation. The amount of alpha-sarcin variant produced in the system was estimated to be 1.2 mg/l of culture.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of chick liver HMG 2a cDNA and developmental expression of HMG 2a mRNA.

The cDNA clone encoding HMG 2a of chick liver was isolated from a lambda gt11 expression library using polyclonal antibodies. DNA sequence analysis revealed an open reading frame of 201 amino acids. Comparison of the nucleotide sequences of cDNA coding for chick liver HMG 2a with pig thymus HMG 2 and human monocytic leukemia cell HMG 2 showed 70% homology. 2.0 kb and 1.2 kb mRNAs were found in newly hatched chick liver and decreased during postnatal development of chicks.

Amino Acid Sequence↗

PubMed1992

Involvement of lysosomes in substrate stabilization of tryptophan-2,3-dioxygenase in rat liver.

Administration of tryptophan or hydrocortisone to rats caused a several-fold increase in tryptophan-2,3-dioxygenase activity in the liver. Highly purified lysosomes were isolated from livers of tryptophan- or hydrocortisone-treated animals as well as the control rats. Immunoblotting of lysosomal proteins with anti-tryptophan-2,3-dioxygenase showed 48 kDa band, corresponding to the subunit molecular weight of the enzyme. The relative amount of the immuno-reactive substance in the lysosomes from hydrocortisone-treated rats was 3 times higher than the control while the value in the lysosomes from tryptophan-treated rats was essentially the same as in the control. These results indicate that administration of tryptophan renders cytosolic tryptophan-2,3-dioxygenase less vulnerable to lysosomal uptake and causes an accumulation of the enzyme in the cytosol.

Animals↗

PubMed1992

Differential mechanisms of induction of ornithine decarboxylase in rat intestine by L- and D-amino acids.

A single intragastric administration of glycine, L- and D-alanine, and L-and D-serine into rats resulted in a more than 20-fold stimulation of intestinal mucosal ornithine decarboxylase (ODC) within 4 h. The stimulation of ODC activity was accompanied by an increase in the amount of immunoreactive ODC protein. The induction of ODC by D-amino acids was in all likelihood attributable to an enhanced accumulation of ODC-specific mRNA species as revealed by Northern blot and dot-blot hybridization analyses. However, the induction by glycine and L-amino acids was not explainable by changes of mRNA since the changes in mRNA contents were only marginal. Since the turnover rates of L-serine-induced and D-serine-induced intestinal ODC protein were the same as the non-induced control, we concluded that the induction by glycine and L-amino acids was brought about by an increased efficiency of translation of the ODC message.

Amino Acids↗

PubMed1992

Effects of theophylline on the selective increases in intratumoral blood flow induced by intracarotid infusion of adenosine and adenosine triphosphate in C6 glioma-transplanted rat brains.

We have previously reported that the intracarotid administration of adenosine or adenosine triphosphate (ATP) selectively increased blood flow in intracerebrally transplanted C6 glioma cells in rats, using the hydrogen clearance method. In the present paper, we studied the difference between the effects of adenosine and ATP, using theophylline, a P1 purinoceptor blocker. The selective enhancement of the tumor blood flow by intracarotid administration of adenosine was almost totally inhibited by theophylline. In contrast, the selective enhancement by ATP was shown definitely not to be inhibited by theophylline. Therefore, it is supposed that the selective increase of intratumoral blood flow by the intracarotid infusion of adenosine is closely related to the P1 purinoceptor, and the effect of the intracarotid infusion of ATP is composed not only of the effect as degraded into adenosine but also of the effect of ATP itself.

Adenosine↗

PubMed1992

Ontogeny of DNA synthetic rhythms in chick (Gallus domesticus) liver.

1. The diurnal pattern of DNA synthesis and mitotic activity in neonatal (1-4-day-old) chick liver were investigated under various feeding and lighting regimens. 2. In the meal-fed chicks under the condition of light-dark cycle, DNA synthesis exhibited a 12 hr cycle; the peaks occurring at 9:00 and 21:00. 3. Fasting caused a gradual decrease in the 21:00 peaks. 4. The changes in the lighting regimen to 24 hr continuous lighting also caused a profound change in the DNA-synthetic pattern, suggesting a complex interplay of feeding and lighting regimens in the manifestation of the DNA-synthetic rhythm in neonatal chick liver.

Animals↗

PubMed1992

Effects of L-NMMA and L-NNA on the selective ATP-induced enhancement of intratumoral blood flow.

We studied the effects of NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L-arginine (L-NNA) on the selective ATP and adenosine-induced enhancement of intratumoral blood flow in rats measured by the hydrogen clearance method. Both adenosine and ATP produced a selective enhancement of the intratumoral blood flow. Neither L-NMMA nor L-NNA had a significant effect on either the CBF or the intratumoral blood flow. Adenosine-induced enhancement was not inhibited by L-NMMA or L-NNA. On the other hand, the ATP-induced enhancement was totally inhibited by both L-NMMA and L-NNA. The inhibitory action of L-NMMA against ATP was blocked by L-arginine, but not by D-arginine. It is suggested that the ATP-induced increase of intratumoral blood flow is evoked by nitric oxide synthesized from the endothelium of the intratumoral blood vessels.

Adenosine↗

PubMed1992

Production and polarized secretion of basement membrane components by glomerular epithelial cells.

To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme-linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side.

Animals↗

PubMed1992

Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells.

A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.

Animals↗

PubMed1992

Erythromycin inhibition of lipopolysaccharide-stimulated tumor necrosis factor alpha production by human monocytes in vitro.

The mechanism of clinical effectiveness of low-dose and long-term erythromycin (EM) treatment for diffuse panbronchiolitis, sinobronchial syndrome, and associated otitis media with effusion was investigated by studying the effects of EM on tumor necrosis factor alpha (TNF-alpha) production by cultured human monocytes stimulated with lipopolysaccharide. At concentrations of 0.1 microgram/mL or more, EM inhibited TNF-alpha release from human monocytes stimulated by lipopolysaccharide in a dose-dependent manner. Of the other macrolides tested, roxithromycin, an EM derivative, also showed significant inhibition of TNF-alpha production, whereas josamycin failed to inhibit TNF-alpha release from monocytes. Nonmacrolidic drugs such as minocycline hydrochloride, ofloxacin, or penicillin G had no significant effect on TNF-alpha production. These results suggest that the clinical improvement of chronic respiratory diseases by EM may depend on the suppression of production of inflammatory cytokines such as TNF-alpha.

Anti-Bacterial Agents↗

PubMed1992

Microsurgical and magnetic resonance imaging anatomy of the cerebello-medullary fissure and its application during fourth ventricle surgery.

The cerebellomedullary fissure, the only entrance or exit to the fourth ventricle, is surrounded rostrally by the cerebellar tonsils and the biventral lobules and caudally by the medulla oblongata, the tela choroidea, and the lateral recesses. This fissure is an important route in operations on the fourth ventricle. We studied the microsurgical and magnetic resonance imaging (MRI) anatomy of the fissure by using autopsied normal cerebellum. MRI revealed that the fissure is visible as a slit and is indicated by the enhanced choroid plexus and the flocculus. Oriented by the anatomical information thus obtained, we have surgically treated nine patients with a tumor either in or around the fourth ventricle. Preoperative MRI clearly demonstrated the tumors in relation to the cerebellomedullary fissure. It revealed the precise anatomical location and extension of the tumor, not only its inferior extension but also its lateral one. The MRI findings and microsurgical anatomy of the cerebellomedullary fissure were quite useful for the removal of the tumors in the fourth ventricle.

Aged↗

PubMed1992

Lipophilic soluble protein represents labile protein in rat liver.

Lipophilic proteins, having higher turnover rates than the average of the total soluble protein pool, were separated from soluble rat-liver proteins on alkyl agarose column. The quantitative immunoprecipitation, using the antibody against the lipophilic proteins, showed that the soluble proteins from the liver of fed rats contained about twice as much lipophilic proteins as that of 3-day-starved rats. This result indicates that the lipophilic proteins, at least in part, represent labile protein in rat liver.

Animals↗

PubMed1992

Characterization, self-assembly and reattachment of S layer from Clostridium botulinum type E saroma.

S layer of Clostridium botulinum type E Saroma and its subunits were isolated and characterized for their chemical and morphological properties. The S layer was composed of a number of subunits with apparent molecular weights ranging from about 10 to 150 kDa. The isolated S layer subunits possess the ability to assemble into recrystallized flat sheets in the absence of any supporting layer and to reattach to the cell wall from which they have been removed. Immunoblot analysis using an antiserum against whole cells of the organism showed that 60 kDa and 90 kDa subunits of the S layer were major somatic antigens of the organism. Immunogold-labeling using monospecific antiserum raised to the individual 60 and 90 kDa proteins revealed that both subunits were exposed evenly over the entire cell surface. The amino acid compositions of both subunits showed that aspartate and glutamate were predominant whereas cystine and methionine were poor. The amino acid composition and acidic property of the two subunits of the S layer agree well with the results obtained from the S layers of other bacterial species as well as other pathogenic clostridia.

Amino Acid Sequence↗