Biomedical subjects

T Akaike

Publications and source records attributed to T Akaike.

At least 73 records · Page 4Linked to original sources

PubMed2000

Inhibition of nitric oxide synthesis in primary cultured mouse hepatocytes by alpha-lipoic acid.

Recent work shows that septic or endotoxic shock is associated with lipopolysaccharide and cytokine mixture-induced nitric oxide (NO) synthesis in liver. Here we found that DL-alpha-lipoic acid inhibited but other thiol-containing antioxidants such as glutathione and N-acetylcysteine enhanced lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis in hepatocytes. The inhibitory action of alpha-lipoic acid on hepatocyte NO synthesis was as potent as that of NG-monomethyl-L-arginine without obvious cytotoxicity. Deletion by diethylmaleate or inhibition by buthionine sulfoximine of intracellular glutathione caused a significant decrease in hepatocyte NO synthesis, implying that increased intracellular reduced glutathione levels could not be the reason for alpha-lipoic acid inhibited NO synthesis. alpha-Lipoic acid inhibition of NO synthesis seems to be from alpha-lipoic acid improved carbohydrate metabolism in hepatocytes. Since alpha-lipoic acid is an essential compound existing naturally in physiological systems, it may serve as both a research and therapeutic agent for sepsis.

Animals↗

PubMed2000

Photocrosslinkable chitosan as a biological adhesive.

A photocrosslinkable chitosan to which both azide and lactose moieties were introduced (Az-CH-LA) was prepared as a biological adhesive for soft tissues and its effectiveness was compared with that of fibrin glue. Introduction of the lactose moieties resulted in a much more water-soluble chitosan at neutral pH. Application of ultraviolet light (UV) irradiation to photocrosslinkable Az-CH-LA produced an insoluble hydrogel within 60 s. This hydrogel firmly adhered two pieces of sliced ham with each other, depending upon the Az-CH-LA concentration. The binding strength of the chitosan hydrogel prepared from 30-50 mg/mL of Az-CH-LA was similar to that of fibrin glue. Compared to the fibrin glue, the chitosan hydrogel more effectively sealed air leakage from pinholes on isolated small intestine and aorta and from incisions on isolated trachea. Neither Az-CH-LA nor its hydrogel showed any cytotoxicity in cell culture tests of human skin fibroblasts, coronary endothelial cells, and smooth muscle cells. Furthermore, all mice studied survived for at least 1 month after implantation of 200 microL of photocrosslinked chitosan gel and intraperitoneal administration of up to 1 mL of 30 mg/mL of Az-CH-LA solution. These results suggest that the photocrosslinkable chitosan developed here has the potential of serving as a new tissue adhesive in medical use.

Animals↗

PubMed2000

Heparin-carrying polystyrene to mediate cellular attachment and growth via interaction with growth factors.

Various sugar-carrying polystyrenes (PSs), which consist of synthetic styrene and sugar moieties, are glycoconjugates that are able to attach to polymeric surfaces. Heparin-carrying PS (HCPS) is especially able to retain the binding of heparin-binding growth factors (GFs) such as vascular endothelial GF 165 (VEGF(165)) or fibroblast GF 2 (FGF-2). Human skin fibroblast cells, human coronary smooth muscle cells, and human coronary endothelial cells have good adherence to the HCPS-coated plate. The growth rate of fibroblast cells on HCPS-coated plates is higher than or comparable to fibronectin-coated, gelatin-coated, or tissue culture treated plates, and the HCPS coating inhibits the growth of smooth muscle cells. On the other hand, the growth rate of endothelial cells on HCPS-coated plates in the presence of either VEGF(165) or FGF-2 is comparable to that on fibronectin-coated, gelatin-coated, and tissue culture treated plates. Endothelial cells grow at a higher rate on HCPS-coated plates retained with either VEGF(165) or FGF-2 than on the other coated plates. These results indicate that growth of various cells can be controlled by the HCPS coating, thereby retaining the bioactivity of molecules such as heparin-binding GFs. Thus, HCPS-coated surfaces control selective growth of various cells.

Biocompatible Materials↗

PubMed2000

Generation of lipid peroxyl radicals from edible oils and their biological activities: a need for consideration for anti-radical components and purification processing.

Lipid hydroperoxides (LOOH or oxidized oils) are known as unfavorable food components. Molecular details of the fate and mechanisms of LOOH to exert adverse effects in vivo are, however, little understood. In the present study, we demonstrated that LOOH generated alkylperoxyl radical (LOO*) after reaction with various heme compounds such as myoglobin, cytochrome c, hemin, hematin, etc., but little formation of other radical species was noticed such as L* or LO*. It was also shown that LOO* thus formed exhibits cytotoxicity and caused DNA damages including strand breakage and abasic site formation. This highly toxic LOO* is effectively scavenged by hot water extracts of vegetable (soup), flavonoids, polyphenols as well as tocopherols. Another important finding is that crude vegetable oils are rich in potent-LOO* scavenging activity, which exhibits potent anti-oxidant activity as well; whereas highly purified oils are scanty in such components and LOO* scavenging activity. These findings imply that a considerate processing in the refining of oils should be needed to retain such potent endogenous anti-oxidative radical scavenging-components.

Anti-Bacterial Agents↗

PubMed2000

Inhibition of sequence-specific protein-DNA interaction and restriction endonuclease cleavage via triplex stabilization by poly(L-lysine)-graft-dextran copolymer.

Triplex stabilization by poly(L-lysine)-graft-dextran copolymer within a mammalian gene promoter inhibits the DNA binding activity of nuclear proteins from HeLa cells as well as restriction endonuclease cleavage at physiological pH and ionic conditions in vitro. Electrophoretic mobility shift assays using a 30-mer homopurine-homopyrimidine stretch (located between -170 and -141 bp) of rat alpha 1 (I) collagen gene promoter reveal that the copolymer, at its wide range of charge ratio with DNA, stabilizes triplex DNA and enhances triplex-specific inhibition of the protein-DNA interaction. When the triplex-forming region (located between -165 and -146 bp) of the promoter is engineered at the Bam H1 and Pst 1 sites of a plasmid DNA, copolymer-mediated triplex stabilization also remarkably competes endonuclease activity of BamH1. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of spermine and benzo[e]pyridoindole. Our results indicate that the copolymer, regardless of the length of the target duplex, stabilizes triplexes for significant inhibition of protein-DNA interaction and endonuclease activity. Since stable triplex formation within a short region out of a long native duplex is a prerequisite to confer the therapeutic potential of antigene strategy, triplex stabilization on a long target duplex and inhibition of nuclear protein-DNA interaction may open the possible in vivo applicability of the copolymer.

Animals↗

PubMed2000

Nitric oxide and virus infection.

Nitric oxide (NO) has complex and diverse functions in physiological and pathophysiological phenomena. The mechanisms of many events induced by NO are now well defined, so that a fundamental understanding of NO biology is almost established. Accumulated evidence suggests that NO and oxygen radicals such as superoxide are key molecules in the pathogenesis of various infectious diseases. NO biosynthesis, particularly through expression of an inducible NO synthase (iNOS), occurs in a variety of microbial infections. Although antimicrobial activity of NO is appreciated for bacteria and protozoa, NO has opposing effects in virus infections such as influenza virus pneumonia and certain other neurotropic virus infections. iNOS produces an excessive amount of NO for long periods, which allows generation of a highly reactive nitrogen oxide species, peroxynitrite, via a radical coupling reaction of NO with superoxide. Thus, peroxynitrite causes oxidative tissue injury through potent oxidation and nitration reactions of various biomolecules. NO also appears to affect a host's immune response, with immunopathological consequences. For example, overproduction of NO in virus infections in mice is reported to suppress type 1 helper T-cell-dependent immune responses, leading to type 2 helper T-cell-biased immunological host responses. Thus, NO may be a host response modulator rather than a simple antiviral agent. The unique biological properties of NO are further illustrated by our recent data suggesting that viral mutation and evolution may be accelerated by NO-induced oxidative stress. Here, we discuss these multiple roles of NO in pathogenesis of virus infections as related to both non-specific inflammatory responses and immunological host reactions modulated by NO during infections in vivo.

Humans↗

PubMed2000

Role of nitric oxide and superoxide in acute cardiac allograft rejection in rats.

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.

Acute Disease↗

PubMed2000

Design of a temporally and spatially controlled drug delivery system for the treatment of liver diseases in mice.

Strict regulation of the distribution and degradation kinetics is the ultimate aim of drug delivery system. Regulation of drug delivery would increase the therapeutic efficacy and decrease the potential side effects. We encapsulated and used Z-Asp, a caspase inhibitor in poly-N-p-vinylbenzyl-D-lactonamide (PVLA) coated-poly (L-lactic acid) (PLA)-nanospheres in a mouse model of acute hepatitis. These nanospheres were internalized and accumulated in hepatocytes both in vitro and in vivo. Encapsulation significantly extended the intracellular retention time of the content in hepatocytes, which increased the bioavailability of the caspase inhibitor. In addition, the therapeutic effect was temporally controllable in vivo by modifying the component of the nanospheres. A cocktail of nanospheres with diverse degradation kinetics showed persistent therapeutic effects in acute hepatitis, and only nanospheres that targeted hepatocytes and controlled degradation rescued mice from lethal hepatic injury. This temporally and spatially controlled drug delivery system could be used in various liver diseases.

Animals↗

PubMed2000

Mechanisms of biological S-nitrosation and its measurement.

Nitric oxide (NO) exhibits multiple biological actions through formation of various oxidized intermediates derived from NO. Among them, nitrosothiol adducts (RS-NOs) with the sulfhydryl moiety of proteins and amino acids appears to be an important species in view of its unique chemical reactivity. Understanding of the biologically relevant S-nitrosation mechanism is essential because RS-NOs seem to be critically involved in modulation of intracellular and intercellular signal transduction, including gene transcription, cell apoptosis, and oxidative stress. RS-NOs have been recently found to be formed efficiently via one-electron oxidation of NO catalyzed by ceruloplasmin, a major copper-containing protein in mammalian plasma. Ceruloplasmin is synthesized mainly by hepatocytes, but it is also expressed by other cells such as macrophages and astrocytes. Once RS-NOs are formed, they function as NO transporters in biological systems, the NO being transferred to different sulfhydryls of various biomolecules. This transfer may be mediated by transnitrosation reactions occurring chemically or enzymatically by a means of specific enzymes such as protein disulfide isomerase. The molecular mechanism of biological S-nitrosation is discussed as related to the important physiological and pathophysiological functions of RS-NOs. Also, RS-NO assays that are being successfully used for detection of biological S-nitrosation are briefly reviewed.

Animals↗

PubMed2000

Triplex formation using ODN conjugates with polycation comb-type copolymer.

Polycation comb-type copolymer that is composed of polylysine backbone and dextran side chains (PLL-g-Dex) has previously been shown to stabilize duplex and triplex DNAs quite effectively. In this study, we have conjugated PLL-g-Dex with oligonucleotides (ODN) aiming to increase the triplex stabilizing efficiency of the copolymer. Here we have demonstrated that the copolymer-TFO conjugates selectively stabilize triplex DNA. Also its potential to form triplex DNA was found to be greater than PLL-g-Dex/ODN mixture.

Base Sequence↗

PubMed2000

Acceleration of DNA strand exchange by polycation comb-type copolymer.

The accelerating effect of cationic substances on DNA strand exchange reaction between 20 bp DNA duplex and its complementary single strand was studied. A comb-type polycationic copolymer which is composed of poly (L-lysine) backbone and dextran graft chain (PLL-g-Dex) and known to stabilize triplex DNA expedites the strand exchange reaction under physiological relevant conditions. Electrostatically small excess of the copolymer increased DNA strand exchange rate by 300-fold while large excess of spermine or cethyltrimethylammonium bromide, cationic detergent known to promote markedly hybridization of complementary DNA strands, showed slight effect. It should be noted that the copolymer promotes the strand exchange reaction while it stabilizes double stranded DNA.

Animals↗

PubMed2000

Enhanced ability of heparin-carrying polystyrene (HCPS) to bind to heparin-binding growth factors and to inhibit growth factor-induced endothelial cell growth.

Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF(165), FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO(4)-heparin) and periodate-oxidized alkaline-degraded heparin (IO(4)-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparins. However, HCPSs did not inhibit the mitogenic activity of VEGF(121), which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin-binding growth factors studied and to inhibit heparin-binding growth factor-induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.

Coronary Vessels↗

PubMed2000

Viral mutation accelerated by nitric oxide production during infection in vivo.

Nitric oxide (NO), superoxide (O(2)(-)), and their reaction product peroxynitrite (ONOO(-)) are generated in excess during a host's response against viral infection, and contribute to viral pathogenesis by promoting oxidative stress and tissue injury. Here we demonstrate that NO and peroxynitrite greatly accelerates the mutation of Sendai virus (SeV), a nonsegmented negative-strand RNA virus, by using green fluorescent protein (GFP) inserted into and expressed by a recombinant SeV (GFP-SeV) as an indicator for mutation. GFP-SeV mutation frequencies were much higher in the wild-type mice than in those lacking inducible NO synthase, suggesting that mutation of the virus in vivo is NO dependent. High levels of NO and NO-mediated oxidative stress were induced by GFP-SeV infection in the lung of the wild-type mice, but not in the iNOS-deficient mice, as evidenced by electron spin resonance spectroscopy and immunohistochemical analysis for nitrotyrosine formation as well as histopathological examination. Furthermore, peroxynitrite, an NO-derived reactive nitrogen intermediate, enhanced viral mutation in vitro. These results indicate that the oxidative stress induced by NO produced during the natural course of viral infection increases mutation, expands the quasispecies spectrum, and facilitates evolution of RNA viruses.

Animals↗

PubMed2000

Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production.

Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO(-)), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO(2) and NH(3) from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 microM ONOO(-), i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO(-) against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO(-)-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 microM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO(-) was also suppressed by the addition of 20 mM NaHCO(3) but not by the addition of 20 mM NH(3). In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO(-) was significantly reduced by the addition of urea or NaHCO(3), as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO(-) scavenger, CO(2)/HCO(3)(-), that defends the bacteria from ONOO(-) cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.

Ammonia↗

PubMed2000

Different vasculoprotective roles of NO synthase isoforms in vascular lesion formation in mice.

NO is known to have several important vasculoprotective actions. Although NO is synthesized by 3 different NO synthase (NOS) isoforms, the vasculoprotective action of individual NOS isoforms remains to be clarified. Permanent ligation of the left common carotid artery was performed in control, endothelial NOS (eNOS) knockout (eNOS-KO), and inducible NOS (iNOS) knockout (iNOS-KO) mice. Four weeks after the procedure, neointimal formation and reduction of cross-sectional vascular area (constrictive remodeling) were noted in the left carotid artery. In the eNOS-KO mice, the extent of neointimal formation was significantly larger than in the control or iNOS-KO mice, whereas the extent of vascular remodeling was the highest in the iNOS-KO mice compared with other 2 strains. Antiplatelet therapy with aspirin or antihypertensive treatment with bunazosin failed to inhibit the accelerated neointimal formation in the eNOS-KO mice. These results indicate that eNOS and iNOS have different vasculoprotective actions against the vascular lesion formation caused by blood flow disruption in vivo: NO derived from eNOS inhibits neointimal formation, whereas NO derived from iNOS suppresses the development of constrictive remodeling.

Adrenergic alpha-Antagonists↗

PubMed2000

Growth of L929 cells on polymeric films prepared by Langmuir-Blodgett and casting methods.

The growth and spreading of fibroblast, L929 cells, on various polymeric films prepared by the Langmuir-Blodgett (LB) and casting methods were investigated. L929 cells, which were cultivated on collagen and synthetic polymeric films prepared by the LB method, adhered and spread much more than those on synthetic films prepared by the casting method. This is explained by the fact that cell growth and cell spreading are suitable for L929 cells on the films having serum proteins that contain a high alpha-helix content, because LB films adsorbed those serum proteins estimated from the circular dichroism measurements of the films immersed in cell culture medium. An exponential relationship was observed from the plot of the cell density vs root mean square of roughness of the films, which is estimated by atomic force microscopy, whereas a linear relationship was observed from the plot of the spreading ratio vs the root mean square of roughness. It is suggested that the correlation between the cell growth or spreading ratio and surface roughness of the films where L929 cells were cultivated is considered to be more important than the correlation between the cell growth or spreading ratio and the contact angle of the films.

Animals↗

PubMed2000

Functional evaluation of poly-(N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-4]-D-gluconamide)(PVLA) as a liver specific carrier.

Hepatocytes express the specific C-type lectin, asialoglycoprotein (ASGP) receptor, on the surface to remove the ligand-bearing proteins from circulation. The specific expression and ligand specificity are thought to be the ideal characters for the target of drug or gene delivery. Various galactose-bearing molecules were synthesized for this purpose. However, the biological or functional interaction of these molecules with the ASGP receptor still remains to be elucidated. In this study. we evaluated the functional ability of synthetic galactose polymer ligand, poly-(N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-4]-D-gluconamide) (PVLA), to interact with recombinant ASGP receptors using mouse ASGP receptor (mouse hepatic lectin; MHL) gene-transfected CHO cells. PVLA-coated beads bound to and were endocytosed by the whole (MHL-1/-2) ASGP receptor-expressing CHO cells like hepatocytes while PVMA (poly-(N-p-vinylbenzyl-O-beta-D-glucopyranosyl-[1-4]-D-gluconamide) did not. Interestingly, PVLA-coated beads were also endocytosed by either MHL-1 or MHL-2 alone expressing cells, which are known to be incapable of endocytosing natural ligands. In addition, the endocytosis of PVLA-coated beads by MHL-expressing CHO cells or primary hepatocytes was inhibited only by soluble PVLA but not by the same galactose molecular concentration of soluble asialofetuin. Furthermore, PVLA-coated beads were endocytosed by primary hepatocyte to a significantly higher degree than asialofetuin-coated beads in vitro. These results suggest that PVLA has higher affinity to the ASGP receptor than the natural ligands in blood. Consistently, it was demonstrated that intravenously injected FITC-labeled PVLA but not PVMA drastically accumulated in parenchymal cells of the liver in vivo. Taken together, PVLA exhibiting higher affinity with hepatocytes than natural ligands is thought to be an attractive and practical carrier-ligand for liver targeting.

Animals↗

PubMed2000

Enhanced effect of sulfonylurea (SU) in copolymer comprising a sugar moiety and SU derivative as double ligands on insulin secretion from MIN6 cells.

Copolymers composed of sulfonylurea (SU) as an antagonist of ATP-sensitive K+ channel and sugar moieties as double ligands were synthesized. Insulin secretion from MIN6 cells (insulinoma cell line) in contact with the copolymers was evaluated. MIN6 cells attached to the poly(N-p-vinylbenzyl-D-maltonamide-co-SU) [P(VMA-co-SU)]-coated dishes were in the more aggregated form as compared to other polymer-coated surfaces. By introducing SU into the sugar bearing homopolymer, an enhanced effect on insulin secretion from MIN6 cells was observed due to the specific interaction between SU ligands and SU receptors on the beta-cell membrane. P(VMA-co-SU) composed of SU and non-reducing glucose moieties demonstrated enhanced insulin secretion from MIN6 cells and faster proliferation of MIN6 cells as compared to poly(N-p-vinylbenzyl-D-lactonamide-co-SU) [P(VLA-co-SU)] probably owing to the glucose transporters presence on the MIN6 cell membrane. Insulin secretion from MIN6 cells pretreated with diazoxide as an agonist of ATP-sensitive K+ channel was suppressed.

Adenosine Triphosphate↗