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T Akaike

Publications and source records attributed to T Akaike.

At least 127 records · Page 7Linked to original sources
PubMed1998

Comb-type copolymer: stabilization of triplex DNA and possible application in antigene strategy.

By employing a reductive amination reaction between the epsilon-amino groups of poly(L-lysine) (PLL) and the reductive ends of the hydrophilic dextran (Dex) side chain, we have prepared different comb-type copolymers which varied in the degree of grafting and the length of the hydrophilic Dex chains. The resulting copolymers, poly(L-lysine)-graft-dextran (PLL-g-Dex), were tested for their ability to stabilize triplex DNA in vitro under physiologically relevant conditions. Thermal denaturation (UV-Tm) and circular dichroism experiments revealed that the graft copolymer with the higher degree of grafting of long Dex chains significantly increased the thermal stability of triplex structure of poly(dA). 2poly(dT) by more than 50 degreesC without affecting the transition between triplex and single-stranded DNA or the native structure of DNA. Of importance is that when triplex formation involving a 30-mer target duplex from rat alpha1 (I) collagen promoter was analyzed by an in vitro electrophoretic mobility shift assay, the graft copolymer also remarkably diminished potassium inhibition of the purine motif triplex formation up to 200 mM as well as pH-dependence of the pyrimidine motif triplex formation. Moreover the triplex-stabilizing efficiency of the copolymer was significantly higher than that of other oligocations like spermine and spermidine. We suggest that a molecular design of comb-type copolymers consisting of various types of polycation backbones (e.g., PLL) grafted with different hydrophilic side chains (e.g., Dex) is a novel strategy to create efficient triplex stabilizers that will certainly shed light on possible in vivo application of the antigene strategy.

DNA↗
PubMed1998

Terplex DNA delivery system as a gene carrier.

PURPOSE: To characterize the physical and biochemical properties of the DNA terplex delivery system, which has previously been shown to deliver and express pSV-beta-gal plasmid efficiently in cultured smooth muscle cells (SMC) (1). METHODS: Atomic force microscopy (AFM), zeta-potential measurement (ZP), gel electrophoresis (GE), circular dichroism (CD), fluorescence quenching and 1H-NMR spectrometry were used. RESULTS: AFM showed that the plasmid DNA of about 600 nm long in its extended state was condensed to the size of about 100 nm by terplex formation. The DNA condensing effect of the terplex system was as good as unmodified PLL, as shown by an ethidium bromide displacement assay. Zeta-potential measurement showed that the terplex system exerts a slightly positive surface charge (+2 mV) at a 1:1:1 weight ratio of DNA:LDL:stearyl-PLL, which showed the best transfection efficiency on SMC. GE indicated that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-PLL, indicating that the surface charge of the terplex system became more positive as more stearyl-PLL was added. Results from CD showed that there was no significant changes in tertiary structure of plasmid DNA from the terplex formation. Presence of strong hydrophobic interaction between stearyl-PLL and LDL was confirmed by 1H-NMR, where about a 30% decrease in epsilon-methylene peak of PLL backbone was observed when stearyl-PLL was mixed with LDL, but this phenomenon was not observed when unmodified PLL was used. CONCLUSIONS: Our results indicate that the plasmid DNA, when formulated with the stearyl-PLL and LDL, forms a stable and hydrophobicity/charge balanced terplex system of optimal size for efficient cellular uptake and the DNA is still intact after the terplex formation. This information is expected to be utilized for the development of much improved transfection vectors for in vivo gene therapy.

Animals↗
PubMed1998

Protective effect of linoleic acid on IFN gamma-induced cellular injury in primary culture hepatocytes.

We have previously demonstrated that treatment of hepatocytes with IFN gamma results a series of cellular injury processes, including DNA synthesis arrest, membrane breakage and apoptosis. In the present work, we show that IFN gamma suppresses cellular respiration and protein synthesis in hepatocytes, and that cellular respiration suppression is an early event in the IFN gamma-induced cellular injuries. Polyunsaturated fatty acids (PUFAs) increased cellular respiration of hepatocytes, but only linoleic acid showed some protective effect against IFN gamma-induced cellular respiration suppression. Linoleic acid also reduced other IFN gamma-mediated cellular injuries, including membrane breakage and protein synthesis inhibition. Like linoleic acid, fetal bovine serum also inhibited IFN gamma-induced cellular damage. Increased NAD levels were found in both IFN gamma-treated and non-treated hepatocytes following the addition of PUFAs, but clofibrate, a peroxisome proliferator, bromophenacyl bromide (BPB), an inhibitor of phospholipase, nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, and arachidonic acid, a metabolite of linoleic acid, did not inhibit IFN gamma-induced cellular injury. In addition, the combination of linoleic acid and IFN gamma induced nitric oxide (NO) synthesis in hepatocytes. These results suggest that fatty acid may play an important role in liver homeostasis during chronic inflammatory states and sepsis.

Animals↗
PubMed1998

Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues.

The objective of this study was to investigate the molecular weight (MW) and time-dependence of the phenomenon termed "the enhanced permeability and retention" (EPR) effect in solid tumor, in particular to determine and define the early phase accumulation of macromolecules in tumor and normal tissues and the relationship between blood concentration and tissue clearance. As a model, radioiodinated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers of MW ranging from 4.5 K to 800 K were administered i.v. to mice bearing sarcoma 180 tumor. Within 10 min all HPMA copolymers accumulated effectively in the tumor regardless of MW (1.0-1.5% of injected dose per g of tumor). However, higher MW copolymers (> 50 K) showed significantly increased tumor accumulation after 6 h, while the lower MW copolymers (< 40 K) were cleared rapidly from tumor tissue due to rapid diffusion back into the bloodstream. Blood clearance was also MW-dependent; the lower MW copolymers displayed rapid clearance, with kidney radioactivity of the copolymers of MW < 20 K representing 24% of injected dose per g kidney at 1 min after i.v. administration. Within 10 min these copolymers passed through the kidney and were excreted in the urine. Higher MW copolymers consistently showed kidney levels of 3-5% dose per g kidney in the early phase with no time-dependent accumulation in kidney. There was also no progressive accumulation in muscle or liver, regardless of polymer MW. These results suggest the "EPR effect" in solid tumor primarily arises from in the difference in clearance rate between the solid tumor and the normal tissues after initial penetration of the polymers into these tissues.

Animals↗
PubMed1998

Involvement of bradykinin generation in intravascular dissemination of Vibrio vulnificus and prevention of invasion by a bradykinin antagonist.

Involvement of bradykinin generation in bacterial invasion was examined by using a gram-negative bacillus, Vibrio vulnificus, which is known to invade the blood circulatory system and cause septicemia. V. vulnificus was injected intraperitoneally (i.p.) into mice with or without bradykinin or a bradykinin (B2 receptor) antagonist. Dissemination of V. vulnificus from peritoneal septic foci to the circulating blood was assessed by counting of viable bacteria in venous blood by use of the colony-forming assay. Intravascular dissemination of V. vulnificus in mice was significantly potentiated by simultaneous injection with bradykinin but was markedly reduced by coadministration with the B2 antagonist D-Arg,[Hyp3, Thi(5,8), D-Phe7]-bradykinin. Furthermore, V. vulnificus lethality was significantly increased when bradykinin was administered simultaneously with the bacillus, whereas it was definitely suppressed by treatment with D-Arg,[Hyp3, Thi(5,8), D-Phe7]-bradykinin. Similarly, ovomacroglobulin, a potent inhibitor of the V. vulnificus protease, showed a strong suppressive effect on the V. vulnificus septicemia. We also confirmed appreciable bradykinin production in the primary septic foci in the mouse peritoneal cavity after i.p. inoculation with V. vulnificus. It is thus concluded that bradykinin generation in infectious foci is critically involved in facilitation of intravascular dissemination of V. vulnificus.

Animals↗
PubMed1998

Therapeutic effect of erythromycin on influenza virus-induced lung injury in mice.

Erythromycin (EM) is an antibiotic with potent antiinflammatory effects that is used for treating chronic lower respiratory tract infections. It has been shown that free radicals, such as the superoxide anion and nitric oxide (NO), are pathogenic molecules in viral disease. Much attention has been given to a critical role of NO in the pathologic events of various inflammatory diseases. In the present study, we evaluated the effects of EM on influenza-virus-induced pneumonia in mice infected with a lethal dose of influenza virus A/Kumamoto/Y5/67 (H2N2). The administration of EM at a dose of 3.3 mg/kg/d (intraperitoneally, from Days 1 to 6 after infection), significantly improved the survival rate of mice infected with influenza virus, and the survival rate of the virus-infected mice at Day 20 after infection increased in a dose-dependent fashion with EM administered to the animals, from 14% among controls to 42% among animals given EM at 1.0 mg/kg/d and 57% among those given EM at 3.3 mg/kg/d. The induction of interferon-gamma (IFN-gamma) in the mouse lung was inhibited by EM treatment on Day 6 after infection. Simultaneously, the number of inflammatory cells recovered in lung lavage fluid 6 d after virus infection was significantly reduced by the treatment with EM. The EM treatment resulted in a dose-dependent decrease in the level of nitrite/nitrate (metabolites of NO) in the serum and the NO synthase (NOS)-inducting potential in the lungs of the virus-infected mice. These results indicate that EM may have substantial therapeutic value for various acute inflammatory disorders such as influenza-virus-induced pneumonia, by inhibiting inflammatory-cell responses and suppressing NO overproduction in the lung.

Animals↗
PubMed1998

Improved nitric oxide detection using 2,3-diaminonaphthalene and its application to the evaluation of novel nitric oxide synthase inhibitors.

A specific and sensitive detection method for nitric oxide (NO) in living cells and tissue culture systems is required in the search for novel NO synthase (NOS) inhibitors. We have improved a fluorometric determination with 2,3-diaminonaphthalene (DAN) by the addition of 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) as an oxidant to form NO2 from NO. This method is 3 times more sensitive than that without PTIO, and is suitable for examining the NOS-inhibitory activity of large numbers of test compounds using a 96-well microplate reader. The improved method was applied to N-monomethyl-L-arginine (L-NMMA) as a known inhibitor and the derivatives of 2-phenyl-1,2-benzisoselenazol-3(2H)-one as teat compounds in order to investigate the effect of these compounds on NO production from activated rat aortic smooth muscle cells. The results obtained indicate that this method is suitable for the rapid assay of large numbers of test compounds.

2-Naphthylamine↗
PubMed1998

Human matrix metalloprotease activation by insults of bacterial infection involving proteases and free radicals.

We found that human matrix metalloproteases (MMPs) may be processed from their proenzyme forms (proMMP) to their active forms by two new and unique mechanisms: Firstly, by bacterial proteases such as Pseudomonas elastase and Vibrio cholerae protease, which cleave off the N-terminal autoinhibitory domain (so-called cysteine switch) from proMMPs. The second mechanism depends on free radical generation by activated polymorphonuclear leukocytes (PMNs). In this case, peroxynitrite (ONOO-) or nitrogen dioxide radical (.NO2), the reaction products of either superoxide (O2.-) or molecular oxygen (O2) and nitric oxide (.NO), are the key reactants. Both O2.- and .NO are generated by activated macrophages and PMNs as a result of immunologic responses involving various proinflammatory cytokines. .NO2 or ONOO- seems to interact with a single cysteine residue in the propeptide autoinhibitory domain, or so-called cysteine switch of proMMPs, thus transforming proMMPs into their active conformation. Furthermore, reactive oxygen species are known to inactivate the alpha1-protease inhibitor (alpha1-PI), a potent neutrophil elastase inhibitor in plasma. In addition, we found that such radicals activate MMPs which degrade and inactivate alpha1-PI by proteolysis. Thus, the activation of MMPs, accompanied by the inactivation of alpha1-PI, will bring about enhanced proteolytic damage to the matrix tissues of the infected sites by both MMPs and elastase.

Bacterial Proteins↗
PubMed1998

Free radicals in viral pathogenesis: molecular mechanisms involving superoxide and NO.

The importance of free radical molecular species in the pathogenesis of various viral diseases has been increasingly recognized in recent years. Oxygen radicals such as superoxide (O2-) and hydroxyl radical (.OH) have been implicated as possible pathogenic molecules in viral disease pathogenesis. Much attention has been given to another simple inorganic radical [nitric oxide (NO)] in the host's defense mechanism and pathogenesis of virus infection. The NO synthesis pathway, in particular, the inducible isoform of NO synthase (iNOS), is expressed in different viral diseases via induction of proinflammatory cytokines such as interferon-gamma. iNOS produces an excessive amount of NO for a long time compared with other constitutive isoforms of NOS (i.e., neuronal NOS and endothelial NOS). Recent studies indicate that NO and O2- are produced in excess during the host's defense responses against various intruding microbes. Reactive nitrogen oxide species such as peroxynitrite (ONOO-) and NOx (NO2 and N2O3) are produced in biological systems through the reaction of NO with either O2- or O2. Among these reactive nitrogen species, ONOO- and its biological actions are of considerable interest in that ONOO- causes oxidation and nitration of amino acid residues of proteins and guanine of DNA, lipid peroxidation, and DNA cleavage. Because the ONOO- is formed via a diffusion-limited fast reaction of NO and O2-, it may be a dominant nitrogen oxide species during the host's defense reactions, when both NO and O2- are produced in excess. Thus, understanding the role of NO and oxygen radical generation in virus infections will provide insight into not only viral pathogenesis but also the host-pathogen interaction in microbial infections at a molecular level.

Animals↗
PubMed1998

[Examination for prediction of residual cholesteatoma].

Residual cholesteatoma, recurrent cholesteatoma, and the open cavity problem still remain to be solved in the surgical treatment of middle ear cholesteatoma. To avoid residual cholesteatoma, complete removal of the cholesteatoma matrix is necessary, but it is sometimes difficult to completely remove it at the initial operation. It is important to estimate the amount of residual cholesteatoma and to completely remove it before its enlargement causes several complications. We established some criteria in order to estimate the residual cholesteatoma at the initial operation, and analyzed the relationship between the criteria and the actual residual cholesteatoma at the second operation. We divided the degree of expectation of residual cholesteatoma into 4 groups: 'none', 'low-rate', 'high-rate', and 'certain'. 'Certain' means that we intentionally left a small part of the matrix to avoid inner ear damage during the procedures. Of 200 cholesteatoma patients who had undergone tympanoplasty between 1985 and 1993, we reviewed 118 who had also undergone a second look or a second stage surgical procedure. Residual cholesteatoma had developed in 25 patients. In those patients, 5 had two pieces of residual cholesteatoma. The incidence of residuum was 0%, 16.5%, 30% and 83.6% in the 'none', 'low-rate', 'high-rate', and 'certain' groups, respectively. The criteria were statistically analyzed and are discussed from the viewpoint of several parameters which would be expected to influence the development of residual cholesteatoma. The analysis disclosed that only one factor influenced the development. The factor was whether the operator could or could not maintain the continuity of the cholesteatoma matrix during the procedure. According to the result, we revised the criterion. The new criterion was considered to be valuable when residual cholesteatoma was predicted, and when adaptation of an exploratory operation was decided.

Adolescent↗
PubMed1998

Nitric oxide and oxygen radicals in infection, inflammation, and cancer.

In recent years, accumulated evidence indicates that free radical species and nitric oxide (NO) or its derivatives are the key denominators in carcinogenesis. Our present topics discussed in this article will focus on the biological significance of free radical generation induced by viral and bacterial infections. In influenza virus infection in mice, the level of xanthine oxidase (XO) at the infected sites was elevated to a great extent. The timing of paralleled induction of XO with that of inducible NO synthase (iNOS) indicates efficient simultaneous reaction: NO + O2*- --> ONOO- (peroxynitrite). Peroxynitrite formation was identified by immunostaining of nitrotyrosine at the local site of infected organs. Peroxynitrite exhibits unique chemical reactivities such as protein nitration, DNA-strand breakage, guanine nitration, etc., which may then bring about not only cytotoxic effect but also mutagenesis. Numbers of evidence in vitro and in vivo show that treatment with chemical carcinogens such as carbon tetrachloride and heterocyclic amines also generated superoxide. The chronic inflammatory reactions, e.g., zymosan- and silica-induced granuloma, revealed very similar free radical generation in vivo. In addition, most experimental solid tumors have elevated levels of iNOS in the tumor tissue, and NO thus generated facilitates vascular permeability, which accelerates nutritional supply to the tumor tissue and hence sustains the rapid tumor growth. These circumstantial evidences suggest that inflammatory responses induced by various pathogens would accelerate mutagenesis as well as tissue damage, whereas NO also sustains more effectively solid tumor growth when normal cells are transformed to tumor or carcinoma cells by the host-derived free radical species.

Animals↗
PubMed1998

Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis.

A diet high in fat and iron is known as a risk factor in cancer epidemiology. However, the details of the molecular mechanism remains to be elucidated. We examined the possible implication of lipid peroxyl radicals generated from fatty acids and heme-iron in DNA damage, and hence in the possibility of colon cancer. F344 female rats were given N-nitroso-N-methylurea six times during a 2-week period and then fed diets containing different amounts of safflower oil and hemoglobin (rich in iron) for 36 weeks; the occurrence of colon cancer was determined by H&E staining. In this animal model, simultaneous feeding of a fat diet and heme-iron produced a significant increase (P < 0.05) in the incidence of colon cancer compared with a diet without hemoglobin. Electron paramagnetic resonance and chemiluminescence studies revealed that oxidized refined vegetable oils, particularly safflower oil, readily generated lipid peroxyl radicals in the presence of various heme compounds, and the peroxyl radicals did effectively cleave DNA. Unpurified native vegetable oils contain a high amount of peroxyl radical scavengers, whereas conventional refining processes seem to reduce the levels of many valuable anti-peroxyl radical compounds abundant in plant seeds. In conclusion, lipid peroxides and heme components generate peroxyl radical species that exert DNA-cleaving activity. A plausible explanation is that lipid peroxyl radicals thus generated, which originated from routine dietary components such as fat and red meat, may contribute, at least in part, to the high incidence of colon cancer.

Animals↗
PubMed1997

Nitric oxide is not involved in lipopolysaccharide and cytokine mixture-induced cellular injuries in primary cultured hepatocytes.

Nitric oxide (NO) from artificial NO donors induces cell death through complete inhibition of mitochondrial respiration of hepatocytes. Treatment of hepatocytes with lipopolysaccharide (LPS) and cytokine mixture (interferon gamma and tumor necrosis factor alpha) not only results in NO production but also causes cellular respiration suppression and cell death in hepatocytes. NG-monomethyl-L-arginine, a specific inhibitor of nitric oxide synthase, inhibits hepatocyte NO synthesis but cannot prevent hepatocytes from LPS and cytokine mixture-induced cellular injuries. Similarly, some metabolic intermediates capable of inhibiting hepatocyte NO synthesis cannot block LPS and cytokine mixture-mediated cellular injuries in hepatocytes. These results imply that lipopolysaccharide and cytokine mixture-induced cellular injuries and NO syntheses are parallel events, NO is not involved in LPS and cytokine mixture-induced cell damage.

Animals↗
PubMed1997

TNF-alpha bifunctionally induces proliferation in primary hepatocytes: role of cell anchorage and spreading.

In the absence of a growth factor or an appropriate extracellular matrix (ECM), cells are arrested in the G0/G1 phase. In this report, we demonstrate the evidence that TNF-alpha induced DNA synthesis of primary mouse hepatocytes in vitro by activating two distinct pathways. TNF-alpha induced drastic spreading of hepatocytes on hydrophobic plastic, while the adhesion was not influenced. The effect was time and dose dependent. The cell spreading was accompanied by the phosphorylation of paxillin, indicating the stimulation of focal adhesion molecules. TNF-alpha-induced spreading of hepatocytes was not transient, and kinetic analysis and morphologic observation suggest that the effect was different from epidermal growth factor- or hepatocyte growth factor-induced transient hepatocyte spreading. TNF-alpha-induced hepatocyte spreading was blocked by cytochalasin D, Arg-Gly-Asp peptides, cycloheximide, or anti-integrin beta1 Ab. Results of competitive PCR for ECM proteins demonstrated that TNF-alpha increased the expression of laminin alpha3 and gamma1 chains in hepatocytes. These data suggested that TNF-alpha induced cell anchorage for hepatocytes by up-regulating ECM production. More importantly, TNF-alpha, but neither epidermal growth factor nor hepatocyte growth factor, induced DNA synthesis following the spreading in primary hepatocytes on hydrophobic plastic, while mere cell spreading on collagen did not induce DNA synthesis. The DNA synthesis was blocked by the inhibition of either cell spreading or DNA polymerase, demonstrating that TNF-alpha induced DNA synthesis in primary hepatocytes by activating two distinct pathways, i.e., forming the scaffold and inducing growth signals. Taken together, TNF-alpha bifunctionally regulates the proliferation of primary hepatocytes, serving as both an ECM inducer and a growth factor.

Animals↗
PubMed1997

Inhibition of lipopolysaccharide and cytokine mixture-mediated hepatocyte nitric oxide synthesis by dimethyl sulfoxide.

Treatment and pretreatment of hepatocytes with 2% dimethyl sulfoxide (DMSO) inhibited lipopolysaccharide and cytokine mixture (LPS/CM)-mediated NO synthesis in hepatocytes without any obvious effects on cell viability. DMSO at concentrations of 0.5-4% stimulated DNA replication and increased albumin secretion in LPS/CM-treated hepatocytes. Genisein, a inhibitor of protein tyrosine kinase (PTK), inhibited LPS/CM-mediated NO synthesis in hepatocytes. These results suggest that PTK is critical for hepatocyte NO synthesis, and DMSO-inhibited NO synthesis may be associated with prevention of LPS/CM-induced PTK activation in hepatocytes.

Animals↗
PubMed1997

Artificial glycopolypeptide conjugates: simple synthesis of lactose- and N,N'-diacetylchitobiose-substituted poly(L-glutamic acid)s through N-beta-glycoside linkages and their interaction with lectins.

Poly(glutamic acid)s carrying lactose- and N,N'-diacetylchitobiose residues were synthesized via a simple two-step procedure. The oligosaccharides were treated with ammonium hydrogen carbonate and the resulting N-beta-glycosylamine was coupled with pendant carboxyl groups of poly(L-glutamic acid) in the presence of a mixture of benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 1-hydroxybenzotriazole (HOBt) in dimethyl sulfoxide. The oligosaccharide incorporated as pendant N-glycosides was estimated by 1H-NMR spectroscopy to be 30 mol% (or 45 wt%) for lactose and 27 mol% (46 wt%) for chitobiose. The glycopolypeptide carrying N,N'-diacetyl-chitobiose inhibited hemagglutination activity of wheat germ agglutinin (WGA) much more strongly (about 10(6) times) than N,N'-diacetylchitobiose itself. The high activity is due to the cluster or high density effect of the glycopolypeptides.

Glycopeptides↗
PubMed1997

Role of metabolic intermediates in lipopolysaccharide/cytokine-mediated production of nitric oxide in isolated mouse hepatocytes.

Induction of nitric oxide synthase by bacterial endotoxin in vivo can be mimicked by treating cultured hepatocytes with a combination of lipopolysaccharide and cytokines (LPS/cytokines), but the role of LPS/cytokine-induced nitric oxide in hepatocyte glucose metabolism is ambiguous. In this study, intermediary metabolite effects on LPS/cytokine-induced hepatocyte nitric oxide synthesis were examined. Pyruvate, lactate, oxaloacetate, and fumarate all showed some inhibitory effects on hepatocyte nitric oxide synthesis. However, these metabolic intermediates could not improve the mitochondrial respiration of LPS/cytokine-treated hepatocytes. Phosphoenolpyruvate carboxykinase activity (or flux) relating factors, glucocorticoids and cAMP, also blocked LPS/cytokine-induced nitric oxide synthesis. Insulin was much less potent than cAMP and glucocorticoids, and phorbol ester did not show any effect on hepatocyte nitric oxide synthesis. These results suggest that LPS/cytokine-induced nitric oxide synthesis is related, at least partly, to phosphoenolpyruvate carboxykinase activity (or flux) in hepatocytes.

Animals↗