Search PubMed⌕ Search

Biomedical subjects

R Sato

Publications and source records attributed to R Sato.

At least 91 records · Page 5Linked to original sources
PubMed1999

Characterization of cleavage enzymes for sterol regulatory element binding protein in hamster liver microsomes.

Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are the key transcription factors for the regulation of the cellular cholesterol level. To identify proteolytic enzymes for SREBPs, a fluorogenic peptide substrate, MOCAc-GRSVLSFK(Dnp)rr-NH2, was synthesized according to the proposed cleavage site of human SREBP-2. In microsome fractions from hamster liver, we found a peptidase activity inhibitable by the synthetic inhibitor Ac-GRSVL-aldehyde with an IC50 of 40 nM. This peptidase separated into three peaks of approximately 400 kDa, 60 kDa, and 30 kDa (Mp400, Mp60 and Mp30 respectively) upon gel permeation chromatography. Mp30 was purified to apparent homogeneity with an Mr of 32 kDa. The partial amino acid sequence of Mp30 possessed homology to cathepsin B (EC 3.4.22.1). A 109 kDa protein band on SDS-PAGE which corresponded to Mp400 exhibited homology to neprilysin (EC 3.4.24.11) in partial amino acid sequence. These findings suggest several degradative pathways for SREBP in liver microsome membranes.

Amino Acid Sequence↗
PubMed1999

Enhancing effects of salicylate on tonic and phasic block of Na+ channels by class 1 antiarrhythmic agents in the ventricular myocytes and the guinea pig papillary muscle.

OBJECTIVE: To study the interaction between salicylate and class 1 antiarrhythmic agents. METHODS: The effects of salicylate on class 1 antiarrhythmic agent-induced tonic and phasic block of the Na+ current (INa) of ventricular myocytes and the upstroke velocity of the action potential (Vmax) of papillary muscles were examined by both the patch clamp technique and conventional microelectrode techniques. RESULTS: Salicylate enhanced quinidine-induced tonic and phasic block of INa at a holding potential of -100 mV but not at a holding potential of -140 mV; this enhancement was accompanied by a shift of the hinfinity curve in the presence of quinidine in a further hyperpolarized direction, although salicylate alone did not affect INa. Salicylate enhanced the tonic and phasic block of Vmax induced by quinidine, aprindine and disopyramide but had little effect on that induced by procainamide or mexiletine; the enhancing effects were related to the liposolubility of the drugs. CONCLUSIONS: Salicylate enhanced tonic and phasic block of Na+ channels induced by class 1 highly liposoluble antiarrhythmic agents. Based on the modulated receptor hypothesis, it is probable that this enhancement was mediated by an increase in the affinity of Na+ channel blockers with high lipid solubility to the inactivated state channels.

Action Potentials↗
PubMed1999

Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter.

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.

ATP-Binding Cassette Transporters↗
PubMed1999

Enzymatic synthesis of Kdn oligosaccharides by a bacterial alpha-(2-->6)-sialyltransferase.

Synthesis of CMP-deaminoneuraminic acid (CMP-beta-D-Kdn) and its enzymatic transfer reaction using bacterial alpha-(2-->6)-sialyltransferase were examined. CMP-beta-D-Kdn was prepared from methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-beta-D-galacto-2- nonulopyranosonate (2) in 24% overall yield. Enzymatic synthesis of Kdn oligosaccharide with CMP-beta-D-Kdn (10.2 mumol), methyl beta-D-lactosaminide (7, 8.1 mumol) and purified sialyltransferase (80 munits) afforded Kdn-alpha-(2-->6)-Gal-beta-(1-->4)-GlcNAc-beta-1-OMe in 77% yield.

Amino Sugars↗
PubMed1999

The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains.

We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form.

Acute-Phase Proteins↗
PubMed1999

cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN.

Amino Acid Sequence↗
PubMed1999

Quantitative differences in the active-site hydrophobicity of five human glutathione S-transferase isoenzymes: water-soluble carcinogen-selective properties of the neoplastic GSTP1-1 species.

The active-site (the H-site) hydrophobicity of five human glutathione S-transferases (GSTs) was analyzed by application of linear free energy relationships (LFERs) with a series of S-alkylated glutathione inhibitors, GS(CH2)n - 1CH3 (n = 1-14). Distinct linear reltionships were observed in the plots of log Ki (inhibition constant) vs n for the five forms, whereby the Kis varied by three to four orders of magnitude. Mean free enthalpy changes per methylene group (-Delta DeltaG degreess), a measure of H-site hydrophobicity, were in the order M1-1 (4.6 kJ/mol) > A1-1 (3. 9 kJ/mol) > A1-2 (3.8 kJ/mol) > A2-2 (2.8 kJ/mol) > P1-1 (1.6 kJ/mol). The quantitative differences may in part account for the extraordinary broad and overlapping substrate specificities of the Alpha-, Mu-, and Pi-class isoenzymes. In contrast to the Alpha and Mu classes being selective for strongly electrophilic compounds, the neoplastic P1-1 species was indicated to be selective for weakly electrophilic and water-soluble carcinogens such as acrolein and hydroxyalkenals.

Binding Sites↗
PubMed1999

The physiological role of sterol regulatory element-binding protein-2 in cultured human cells.

To clarify the role of the sterol regulatory element-binding protein-2 (SREBP-2), we established cell lines in which human SREBP-2(1-481) could be induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The range of IPTG-induced changes in SREBP-2(1-481) levels in '23-11' cells, one of these cell lines, was almost the same as that of sterol-induced changes in the levels of mature SREBP-2, indicating that IPTG was able to regulate the expression of SREBP-2(1-481) within the normal physiological range in this cell line. Sterols regulate the expression of the LDL receptor, HMG-CoA reductase, squalene synthase and fatty acid synthase in 23-11 cells as they also do in the parental cell line HeLa S3. IPTG increased mRNA levels of the LDL receptor and HMG-CoA reductase but not squalene synthase both in the presence or absence of excess sterols. Fatty acid synthase mRNA was increased 2 h after the IPTG addition in the absence of excess sterol (10% FBS), but was slightly increased 6 h after the IPTG addition in the presence of excess sterols. In the absence of excess sterols, both SREBP-2(1-481) and endogenous mature SREBP-2 exist in the nucleus. This suggests that an increased amount of SREBP-2 over the normal physiological range is required for the regulation of fatty acid synthase. IPTG increased both the surface binding of 125I-LDL and cholesterol biosynthesis from [14C]acetate significantly in a similar time course. In contrast, fatty acid biosynthesis from [14C]acetate was almost unchanged by IPTG during the same incubation period. These results suggest that physiological amounts of SREBP-2 play a key role in the regulation of cholesterol but not fatty acid metabolism.

Cell Line↗
PubMed1999

Acaloleptins A: inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa.

We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem.

Amino Acid Sequence↗
PubMed1999

Binding of phylogenetically distant Bacillus thuringiensis cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's conserved structure in receptor binding.

We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site.

Amino Acid Sequence↗
PubMed1999

Mechanisms of cation permeation in cardiac sodium channel: description by dynamic pore model.

The selective permeability to monovalent metal cations, as well as the relationship between cation permeation and gating kinetics, was investigated for native tetrodotoxin-insensitive Na-channels in guinea pig ventricular myocytes using the whole-cell patch clamp technique. By the measurement of inward unidirectional currents and biionic reversal potentials, we demonstrate that the cardiac Na-channel is substantially permeable to all of the group Ia and IIIa cations tested, with the selectivity sequence Na(+) >/= Li(+) > Tl(+) > K(+) > Rb(+) > Cs(+). Current kinetics was little affected by the permeant cation species and concentrations tested (</=160 mM), suggesting that the permeation process is independent of the gating process in the Na-channel. The permeability ratios determined from biionic reversal potentials were concentration and orientation dependent: the selectivity to Na(+) increased with increasing internal [K(+)] or external [Tl(+)]. The dynamic pore model describing the conformational transition of the Na-channel pore between different selectivity states could account for all the experimental data, whereas conventional static pore models failed to fit the concentration-dependent permeability ratio data. We conclude that the dynamic pore mechanism, independent of the gating machinery, may play an important physiological role in regulating the selective permeability of native Na-channels.

Animals↗
PubMed1999

Lipopolysaccharide-binding protein of Bombyx mori participates in a hemocyte-mediated defense reaction against gram-negative bacteria.

BmLBP is a lipopolysaccharide-binding protein in B. mori and participates in bacterial clearance in vivo. Here, we investigated the function of BmLBP more specifically. More than 90% of injected gram-negative rough strains to which BmLBP binds were removed from the plasma within 30 min post-injection, whereas it required 8h for the clearance of smooth strains to which BmLBP does not bind. Observation of the hemocoel after the injection of Escherichia coli rough strain showed that melanized nodules were formed at 30 min post-injection when the clearance of injected E. coli cells had occurred. Fluorescence microscope observation revealed that E. coli cells were actually trapped in the nodules formed in vivo. Furthermore, plasma pre-treated E. coli rough cells (BmLBP bound) added to hemocytes isolated in vitro caused vigorous hemocyte aggregations with the bacteria, while plasma pre-treated smooth cells did not. The formation of aggregates was inhibited by anti-BmLBP serum pre-treatment, suggesting that BmLBP causes the clearance of bacteria by promoting hemocyte nodule formation.

Journal Article↗
PubMed1999

Mechanism of inhibition of the Na current by tocainide in guinea-pig isolated ventricular cells.

Tocainide blocked the Na current (I(Na)) in ventricular myocytes in either a tonic or a phasic block manner, having a higher affinity for the inactivated state (K(di) = 18 microM) than for the rested state (Kd(rest) = 606 microM). The degree of phasic block was enhanced and the onset of phasic block was faster with an increase in pulse duration as well as at less-negative holding potential (HP). The recovery-time constant from the phasic block of I(Na) induced by tocainide was independent of either the HP or the removal of fast inactivation. After removal of fast inactivation, tocainide showed the pulse-duration dependency of phasic block but not the voltage dependency. These results suggest that tocainide could bind to the inactivated-state receptor through the hydrophilic pathway and leave the receptor through the hydrophobic pathway, producing the tonic and phasic block of I(Na).

Animals↗
PubMed1999

Structure-physicochemical function relationships of soybean beta-conglycinin constituent subunits.

Beta-conglycinin, one of the dominant storage proteins of soybean, has a trimeric structure, being composed of three subunits alpha, alpha', and beta. The alpha and alpha' subunits contain the extension regions in addition to the core regions common to all subunits, which are N-glycosylated. Physicochemical functions of recombinant nonglycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of the alpha and alpha' subunits were examined at pH 7.6 and 3.7 at low (mu = 0.08) and high (mu = 0.5) ionic strengths. Although individual recombinant subunits exhibited different properties at all conditions, there were some consistencies. Surface hydrophobicities and thermal stabilities of the individual subunits were likely to be conferred by their core regions, and the carbohydrate moieties did not contribute to these properties at any conditions examined here. Solubility at mu = 0.08, heat-induced association, and emulsifying ability remarkably depended on the extension regions and the carbohydrate moieties in addition to the structural features of the core regions. These findings indicate that various end products could be produced by the selection of soybean varieties containing beta-conglycinin with different subunit compositions and suggest a direction for a principle of soybean breeding.

Antigens, Plant↗
PubMed1999

Effects of amlodipine on native cardiac Na+ channels and cloned alpha-subunits of cardiac Na+ channels.

The inhibitory effects of amlodipine besilate (CAS 11470-99-6) on the native Na+ current (INa) and cloned human cardiac Na+ channel alpha subunit (hH1) were studied by whole cell patch clamp techniques. Amlodipine produced tonic block of INa in a concentration- and holding potential (HP)-dependent manner with hyperpolarization of H infinity. Amlodipine produced phasic blockade of INa, which was dependent on HP and pulse duration. Amlodipine produced tonic blockade of hH1 in a concentration-dependent manner with 1 : 1 stoichiometry, and phasic blockade of hH1 which was dependent on the pulse duration. Amlodipine blocked INa in a voltage- and frequency-dependent manner via affinity to the resting as well as inactivated conformations of the alpha subunit.

Algorithms↗
PubMed1999

Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine.

1. Aminopyrine N-demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by Lineweaver-Burk plots. CYP2C19 exhibited the highest affinity and a high capacity for the aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the Km for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b5, whereas the activity was not affected by the addition of Mg2+. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.

Aminopyrine↗
PubMed1999

Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip.

The sterol regulatory element-binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix-leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin beta. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin beta in the absence of importin alpha. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2-importin beta complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin beta in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix-leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin beta.

Binding Sites↗