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Biomedical subjects

T Akaike

Publications and source records attributed to T Akaike.

At least 199 records · Page 11Linked to original sources
PubMed1994

Cellular distribution of polymer particles bearing various densities of carbohydrate ligands.

The density effect of carbohydrate-ligands on nanometer-order particles (nanoparticles) upon cellular binding and internalization was investigated. Poly(vinylbenzyl-beta-D-lactonamide) (PVLA), a beta-galactose-carrying styrene homopolymer, was employed as a model ligand for the asialoglycoprotein receptors on hepatocytes. In order to control the surface ligand densities on the particles, PVLA was mixed with poly(vinylbenzyl-D-gluconamide) (PVGA), a PVLA analog without beta-galactose, and their mixtures were used as surface coatings. The particles with low ligand densities associated more with hepatocytes than high ligand density particles. The surface density of the ligand considerably influenced the cellular distribution. Most of the particles bearing high densities of ligands were found inside the cells, whereas particles with low ligand densities were found on the plasma membrane surface of the hepatocytes. These results were indicative of high densities of ligands on the surface requiring hepatocytes to internalize the particles promptly by receptor-mediated endocytosis, while low densities of ligands on the surface was not sufficient to internalize, but allowed particles to bind on the cell surface. These findings enabled us to regulate cellular distributions of particles by controlling ligand density on the surface.

Animals↗
PubMed1994

Receptor-mediated regulation of differentiation and proliferation of hepatocytes by synthetic polymer model of asialoglycoprotein.

Morphology and responses of hepatocytes are investigated using an artificial asialoglycoprotein model polymer--lactose-carrying polystyrene (PVLA) as a culture substratum, especially in focusing on the effect of the surface density of the PVLA substratum. The surface density of PVLA on polystyrene dishes was determined using fluorescein-labeled PVLA as a probe under a fluorescence laser microscope. PVLA-coated surfaces were observed by scanning electron microscope and atomic force microscopies under air and water, which showed that PVLA molecules were adsorbed patchily on low density surfaces and uniformly concentrated all over the dish on high density surfaces. It is suggested from the requirement of the Ca2+ ion, inhibition of galactosyl substances, and localization of receptors that the adhesion of hepatocytes to both low and high PVLA-density surfaces is mediated by galactose-specific interactions between PVLA and asialoglycoprotein receptors. At low PVLA densities (0.07 micrograms cm-2), the hepatocytes were flat and expressed high levels of 3H-thymidine uptake and low levels of bile acid secretion. Contrastingly, at high PVLA densities (1.08 micrograms cm-2), they were round and expressed a low level of 3H-thymidine uptake and a high level of bile acid secretion. The shapes, proliferation, and differentiation of hepatocytes could be regulated by varying the densities of PVLA adsorbed to polystyrene dishes. We assume that there are two recognition mechanisms operating between PVLA and hepatocytes: (1) adhesion through highly concentrated or clustered galactose-specific interaction; and (2) responses in shape, proliferation, and differentiation by PVLA-coating densities.

Animals↗
PubMed1994

Induction of cytochrome P-450s and expression of liver-specific genes in rat primary hepatocytes cultured on different extracellular matrices.

Freshly isolated hepatocytes were cultured on an EHS-gel prepared from EHS-tumor, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), and type I collagen (TIC). Hepatocytes on EHS-gel showed a spherical shape and much more strongly maintained the inducible expression of cytochrome P-450 genes which were lost on PVLA and TIC. Further, the expression of liver-specific genes were maintained on EHS gel at the highest level, and then higher on PVLA than TIC.

Animals↗
PubMed1994

Inflammatory cytokines up-regulate intercellular adhesion molecule-1 expression on primary cultured mouse hepatocytes and T-lymphocyte adhesion.

We investigated the effect of inflammatory cytokines on the intercellular adhesion molecule-1 expression on primary cultured murine hepatocytes. Tumor necrosis factor-alpha, interferon-gamma and interleukin-1 alpha up-regulated the intercellular adhesion molecule-1 expression on hepatocytes in a dose-dependent fashion; however, interleukin-6 did not. On the basis of kinetic analysis, the expression level reached a peak 24 hr after stimulation, and both cycloheximide and actinomycin D inhibited the expression. Furthermore, T lymphocytes bind more to interferon-gamma-stimulated hepatocytes than to unstimulated hepatocytes. The binding was dependent on the concentration of interferon-gamma. The binding was also up-regulated by stimulating T lymphocytes with phorbol myristate acetate. Tumor necrosis factor-alpha and interleukin-1 alpha demonstrated the same effect as interferon-gamma, whereas interleukin-6 did not increase T-lymphocyte adhesion to the hepatocytes. The adhesion induced by interferon-gamma or tumor necrosis factor-alpha was inhibited by antibody against either intercellular adhesion molecule-1 or lymphocyte function-associated antigen-1, a ligand for intercellular adhesion molecule-1, but was not inhibited by CD44 antibodies. These results demonstrate that inflammatory lymphokines enhance the T-lymphocyte adhesion to primary cultured hepatocytes by up-regulating the intercellular adhesion molecule-1 expression on the stimulated hepatocytes by activating the de novo pathway. This mechanism may play an important role in the pathogenesis of hepatitis.

Animals↗
PubMed1993

Effect of microbial and mite proteases on low and high molecular weight kininogens. Generation of kinin and inactivation of thiol protease inhibitory activity.

Kinin release from guinea pig plasma high molecular weight kininogen (HMWK) induced by various microbial and mite proteases has been demonstrated previously (Molla, A., Yamamoto, T., Akaike, T., Miyoshi, S., and Maeda, H. (1989) J. Biol. Chem. 264, 10589-10594; Maruo, K., Akaike, T., Matsumura, Y., Kohmoto, S., Inada, Y., Ono, T., Arao, T., and Maeda, H. (1991) Biochim. Biophys. Acta 1074, 62-68). In this paper, we describe the effects of various microbial and mite proteases on low molecular weight kininogen (LMWK) and HMWK from human plasma. A protease from the house dust mite Dermatophagoides farinae (Df-protease) directly liberated kinin from both LMWK and HMWK to a significant degree. The Km, kcat, and kcat/Km values for kinin generation from LMWK were 3.24 microM, 0.61 s-1, and 1.9 x 10(5) M-1 x s-1, respectively, and those for kinin generation from HMWK were 0.56 microM, 0.12 s-1, and 2.1 x 10(5) M-1 x s-1, respectively; kcat/Km values for Df-protease were comparable with that for glandular kallikrein. In contrast, microbial proteases showed only weak kinin-releasing activity from both human plasma kininogens. Four of ten different microbial proteases liberated kinin from LMWK, and only serratial 56-kDa protease released kinin from HMWK. Furthermore, Df-protease markedly inactivated the thiol protease inhibitory activity of LMWK and HMWK, whereas all microbial proteases (as well as the endogenous protease trypsin) did not affect this inhibitory activity of both kininogens from human plasma.

Animals↗
PubMed1993

Biosynthesis of curdlan from culture media containing 13C-labeled glucose as the carbon source.

13C-Labeled curdlans were biosynthesized by Agrobacterium sp. (ATCC 31749) from culture media containing D-(1-13C)glucose, D-(6-13C)glucose, or D-(2-13C)glucose as the carbon source, and their structures were analyzed by 13C NMR spectroscopy. The labeling was mainly found in the original position, that is, C-1, C-6, or C-2, indicating direct polymerization of introduced glucose. In addition, C-3 in curdlan obtained from D-(1-13C)glucose, C-1 in curdlan obtained from D-(6-13C)glucose, and C-1 and C-3 in curdlan obtained from D-(2-13)glucose were labeled. From analysis of this labeling, the biosynthesis of curdlan was interpreted as involving five routes: (1) direct synthesis from glucose; (2) rearrangement (1-13C-->3-13C); and (3) isomerization (6-13C-->1-13C) of cleaved trioses by the Embden-Meyerhof pathway, followed by neogenesis of glucose and formation of curdlan; (4) from fructose 6-phosphate formed in the pentose cycle (2-13C-->1-13C, 3-13C); and (5) neogenesis of glucose from fragments produced in various pathways of glycolysis. The 13C-labeling at C-6 and C-2 in the starting glucoses is well preserved in the C-6 carbon and the C-1 to C-3 carbons, respectively, in the curdlan produced.

Carbon Isotopes↗
PubMed1993

Determination of mouse major asialoglycoprotein receptor cDNA sequence.

Hepatic lectins (asialoglycoprotein receptors) specifically recognize galactose-terminated glycoproteins and mediate endocytosis of these molecules. We now report the cloning and sequence of a cDNA encoding murine asialoglycoprotein receptor. It shows high homology with rat and human major receptor forms designated RHL-1 and HHL-1, respectively. They have many conserved regions, such as a transmembrane region, carbohydrate additional region and carbohydrate related region. According to the homology analysis, we concluded that the clone encodes the mouse major asialoglycoprotein receptor (MHL-1).

Amino Acid Sequence↗
PubMed1993

Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/.NO through a radical reaction.

A labile inorganic free radical, nitric oxide (.NO), is produced by nitric oxide synthase from the substrate L-arginine in various cells and tissues. It acts as an endothelium-derived relaxing factor (EDRF) or as a neurotransmitter in vivo. We investigated the reactivity of stable radical compounds, imidazolineoxyl N-oxides such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), carboxy-PTIO, and carboxymethoxy-PTIO against .NO/EDRF in both chemical and biological systems. By using electron spin resonance (ESR) spectroscopy, imidazolineoxyl N-oxides were found to react with .NO in a stoichiometric manner (PTIO/.NO = 1.0) in a neutral solution (sodium phosphate buffer, pH 7.4) with rate constants of approximately 10(4) M-1 s-1, resulting in the generation of NO2-/NO3- and imidazolineoxyls such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI), carboxy-PTI, or carboxymethoxy-PTI. Furthermore, the effects of imidazolineoxyl N-oxides on acetylcholine- or ATP-induced relaxation of the smooth muscle of rabbit aorta were tested. The vasorelaxations were inhibited by all three imidazolineoxyl N-oxides markedly. The inhibitory effects of carboxy-PTIO was almost 2-fold stronger than those of .NO synthesis inhibitors, N omega-nitro-L-arginine and N omega-monomethyl-L-arginine. Generation of EDRF/.NO was identified by reacting the PTIO in aortic strips and quantitating the reaction product with ESR spectroscopy. Thus, it was clarified that imidazolineoxyl N-oxide antagonize EDRF/.NO via a unique radical-radical reaction with .NO.

Acetylcholine↗
PubMed1993

Cell adhesion onto block copolymer Langmuir-Blodgett films.

The attachment of cells onto the surfaces of various block copolymers fabricated either as well-defined, ordered Langmuir-Blodgett (LB) films, or solvent cast microphase-separated structures was studied. In general, more platelets adhered onto the multilayered LB surface than onto microphase-separated cast surfaces. Scanning electron micrographs of adhered platelets showed extensive morphological changes associated with the LB surface as compared to cast film surfaces. The morphology of adhered hepatocytes was similar for both LB films and cast surfaces. It may be assumed that the surface of a block copolymer LB film does not orient into microdomains, as in the solvent cast surfaces, and only one polymer domain interacts at the interface.

Animals↗
PubMed1993

Role of bradykinin in microbial infection: enhancement of septicemia by microbial proteases and kinin.

Data presented herein will show that bradykinin, microbial proteases which activate the kinin generating cascade, and kininase inhibitors can enhance septicemia by approximately 10 to 100 fold in mice infected intraperitoneally (i.p.) with a strain of bacteria, Pseudomonas aeruginosa 621, which does not usually produce a kinin generating protease. Bacterial spreading was evaluated either in the blood or in the spleen by colony formation on agar plates. Using the P. aeruginosa kaguma strain which produces a large amount of proteases, further experiments were carried out. Results showed that two different protease inhibitors (ovomacroglobulin and a synthetic peptide inhibitor against pseudomonal elastase) as well as a kinin antagonist suppressed bacterial dissemination to 1/10-1/100 of control. Similar results were observed in experiments using Vibrio vulnificus. These data support the hypothesis that microbial proteases and especially bradykinin is responsible for facilitation of microbial dissemination in vivo.

Animals↗
PubMed1993

Design of hepatocyte-specific extracellular matrices for hybrid artificial liver.

For the development of an artificial liver which can assist multiple metabolic functions of liver, the effective cultivation of hepatocytes on substrate as extracellular matrix analogs was examined. Adult rat hepatocytes attached on an asialoglycoprotein model polymer, poly-N-p-vinylbenzyl-D-lactonamide (PLVA), formed anchored multilayer aggregates which had a stable three-dimensional structure when epidermal growth factor (EGF) and insulin were added to the culture medium. The formation of multilayer aggregates was also dependent on the specific cellular attachment mediated by asialoglycoprotein receptor of hepatocyte surface. Cells in the aggregates expressed higher level of albumin secretion and lower proliferative ability than those in monolayer cultures on collagen. It seemed likely that the cells in multilayer aggregates experienced stable differentiated states resembling in vivo status through the forming multilayer aggregates. The culture system described here has useful potentiality for the development of hepatic module systems for a hybrid artificial liver.

Animals↗
PubMed1993

Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis.

Susceptibility of three different strains of Mycobacterium avium complex (MAC), i.e., one strain of M. avium (Mino) and two strains of M. intracellulare (31F093T and KUMS 9007), to nitric oxide (NO) generated by rat alveolar macrophages (M phi) or NO generated chemically by acidification of NO2- was examined in vitro. We also investigated the effects of NO on phagocytosis and superoxide anion (O2-) generation by M phi. The intracellular growth of M. avium Mino was significantly suppressed by NO generated by gamma interferon (IFN-gamma)-stimulated M phi, whereas that of two strains of M. intracellulare (31F093T and KUMS 9007) was not. M. avium Mino was also more susceptible to NO generated chemically by acidification of NO2- than the two M. intracellulare strains. In L-arginine (1 mM)-containing medium, NO release from the M phi assessed by measuring NO2- increased as the concentration of IFN-gamma increased. The enhancing potential of IFN-gamma for NO release became more pronounced when M phi were infected with 31F093T, an NO-resistant strain. A large amount of NO generated by IFN-gamma-stimulated M phi suppressed both phagocytosis and O2- generation by the M phi, especially after infection of the M phi with strain 31F093T. These results indicate that the intracellular growth of MAC is not always inhibited by NO generated by immunologically activated M phi; rather, NO generation induced by infection with an NO-resistant MAC strain suppresses phagocytosis of the M phi, which may allow extracellular spreading of such NO-resistant mycobacteria. Therefore, the pathogenic potential of MAC may be partly attributed to its resistance to NO.

Animals↗
PubMed1993

Pronounced enhancement of .NO-dependent antimicrobial action by an .NO-oxidizing agent, imidazolineoxyl N-oxide.

The antimicrobial action of .NO against Cryptococcus neoformans was investigated by using imidazolineoxyl N-oxide, which we recently reported removes .NO via oxidation (T. Akaike, M. Yoshida, Y. Miyamoto, K. Sato, M. Kohno, K. Sasamoto, K. Miyazaki, S. Ueda, and H. Maeda, Biochemistry 32:827-832, 1993). No appreciable fungicidal activity was observed in neutral .NO solutions. Imidazolineoxyl N-oxide induced or enhanced fungicidal action in neutral or acidic .NO solutions, respectively. Our results provide convincing evidence that .NO is not a microbicidal molecular species.

Anti-Infective Agents↗
PubMed1993

Effect of sulfated polysaccharides on hepatocyte adhesion.

The effects of sulfated polysaccharides on hepatocyte adhesion on the polystyrene dish and fibronectin-coated dish were investigated. Dextran sulfate and synthetic mannopyranan sulfate slightly inhibited hepatocyte adhesion on fibronectin-coated dish, while heparin showed no effect. On the other hand, hepatocyte adhesion on polystyrene dishes was stimulated in the presence of sulfated polysaccharides in the medium.

Animals↗
PubMed1992

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.

Cyclic N-Oxides↗
PubMed1992

A new synthetic hypoglycaemic polysaccharide.

The synthetic (1-->6)-alpha-D-glucopyranan with branching and without branching were tested as a new hypoglycaemic drug. (1-->6)-alpha-D-glucopyranan having an alpha-D-glucopyranosyl branch at the C-3 position (1) showed a remarkable hypoglycaemic activity on i.p. injection to mice. The polysaccharide having both alpha- and beta-glucopyranosyl branches (2) also lowered the blood sugar (glucose) level in mice. On the other hand, the synthetic linear (1-->6)-alpha-D-glucopyranan (3) and alpha-D-glucopyranosyl branched polysaccharide (4) did not have a hypoglycaemic function, indicating that the branching glucose units are essential for the biological activity.

Animals↗