Biomedical subjects

T Akaike

Publications and source records attributed to T Akaike.

At least 19 recordsLinked to original sources

PubMed1999

Nitrosothiol formation catalyzed by ceruloplasmin. Implication for cytoprotective mechanism in vivo.

Ceruloplasmin (CP) is a major multicopper-containing plasma protein that is not only involved in iron metabolism through its ferroxidase activity but also functions as an antioxidant. However, physiological substrates for CP have not been fully identified nor has the role of CP been fully understood. The reaction of nitric oxide (NO) with CP was investigated in view of nitrosothiol (RS-NO) formation. First, formation of heavy metal- or CP-catalyzed RS-NO was examined with physiologically relevant concentrations of NO and various thiol compounds (RSH) such as glutathione (GSH). Among the various heavy metal ions and copper-containing enzymes and proteins examined, only copper ion (Cu(2+)) and CP showed potent RS-NO (S-nitrosoglutathione)-producing activity. Also, RS-NO-forming catalytic activity was evident for CP added exogenously to RAW264 cells expressing inducible NO synthase in culture, but this was not the case for copper ion. Similarly, CP produced endogenously by HepG2 cells showed potent RS-NO-forming activity in the cell culture. One-electron oxidation of NO appears to be operative for RS-NO production via electron transfer from type 1 copper to a cluster of types 2 and 3 copper in CP. Neurological disorders are associated with aceruloplasminemia; besides RS-NO, S-nitrosoglutathione particularly has been shown to have neuroprotective effect against oxidative stress induced by iron overload. Thus, we suggest that CP plays an important catalytic role in RS-NO formation, which may contribute to its potent antioxidant and cytoprotective activities in vivo in mammalian biological systems.

Animals↗

PubMed1999

Free radical generation from heterocyclic amines by cytochrome b5 reductase in the presence of NADH.

We previously reported findings that NADPH/cytochrome P450 reductase can generate superoxide anion radical (O2*-) from heterocyclic amines (HCA) and from many anticancer agents in vitro. Here we present more evidence in which O2*- is generated when recombinant human cytochrome b5 reductase (rh-Cytb5Rd) was incubated with HCAs such as IQ and MeIQ in the presence of NADH in vitro. This indicates that free radical generation by rh-Cytb5Rd in the presence of HCA may add new insight into the damage of DNA in addition to the previously known mechanism: interaction of activated HCA-intermediates to form DNA adduct.

Cytochrome Reductases↗

PubMed1999

Combined effects of both bacteria and gastric juice on pneumonia in mice.

The effects of a combined inoculation of gastric juice and Streptococcus pneumoniae on the lungs of mice was investigated. Survival rates of mice inoculated with bacteria alone, gastric juice alone, and both bacteria and gastric juice were compared over 18 days. Xanthine oxidase (XO) activities in the lung tissues of mice inoculated with bacteria and gastric juice were measured and injected with a free radical scavenger, pyran-superoxide dismutase (pyran-SOD). A high mortality rate was observed in mice inoculated with both gastric juice and Streptococcus pneumoniae (81%). Mice inoculated with either Streptococcus pneumoniae or gastric juice showed a separate mortality rate of up to 10% during 18 days after inoculation. XO activity in the lung tissue of the mice inoculated with both gastric juice and bacteria was higher than in mice inoculated with either of them separately. The high mortality rate in the group inoculated with both two agents was reduced to 25% by the administration of pyran-SOD. XO activity raised by Streptococcus pneumoniae and gastric juice was significantly reduced by pyran-SOD. Thus, we suggest an important role in the combined effects of gastric juice and bacteria on pneumonia.

Animals↗

PubMed1999

IRF-1 is an essential mediator in IFN-gamma-induced cell cycle arrest and apoptosis of primary cultured hepatocytes.

IFN-gamma induces cell cycle arrest and p53-independent apoptosis in primary cultured hepatocytes. However, it is not yet understood what molecules regulate the mechanism. We report here that interferon regulatory factor 1 (IRF-1) is an essential molecule in these phenomena. Hepatocytes from IRF-1-deficient mice were completely resistant to IFN-gamma in apoptosis indicated by three different hallmarks such as LDH release, DNA fragmentation and the activation of caspase-3 family. Caspase-1 expression was little detected in hepatocytes, and constitutive and IFN-gamma-induced mRNA expression of Fas or caspase-3 did not change in between wild type and IRF-1-deficient hepatocytes. Expression of IFN-gamma-inducible caspase, caspase-11, did not change either. Thus, it is unlikely that these molecules directly regulate the mechanisms. Interestingly, IRF-1-deficient hepatocytes were also resistant to IFN-gamma-induced cell cycle arrest despite IFN-gamma-induced cell cycle arrest and apoptosis are regulated by independent pathways. Results by Northern blot analysis showed that IFN-gamma-induced but not constitutive p53 mRNA expression was regulated by IRF-1. In fact, IFN-gamma did not induce cell cycle arrest in p53-deficient hepatocytes. Taken together, IRF-1 mediates IFN-gamma signaling into primary hepatocytes for cell cycle arrest via p53 expression and for apoptosis.

Animals↗

PubMed1999

Role of nitric oxide in pathogenesis of herpes simplex virus encephalitis in rats.

The role of nitric oxide (NO) in the pathogenesis of viral encephalitis was investigated by using an experimental model of herpes simplex virus type 1 (HSV-1) encephalitis in Lewis rats. The expression of inducible NO synthase (iNOS) mRNA determined by Northern blotting was observed first in the olfactory bulb and the brain stem on day 5 after intranasal inoculation of HSV-1, and thereafter iNOS mRNA was detected in other brain regions, i.e., cerebrum and cerebellum. In various parts of the brain, excessive NO production was identified by electron spin resonance spectroscopy. The temporal and spatial patterns of iNOS expression coincided with those of viral propagation, as demonstrated by polymerase chain reaction for HSV-1 gene expression as well as by the plaque-forming assay. Immunohistochemical study determined that iNOS was localized mainly in monocyte-derived macrophages. Treatment of virus-infected animals with the NOS inhibitor Nomega-monomethyl-l-arginine (l-NMMA), but not Nomega-monomethyl-d-arginine, significantly ameliorated not only clinical symptoms such as paralysis and seizures but also mortality. Virus yield from brain tissue was not affected by l-NMMA treatment. It is of interest that increased expression of the antioxidant enzyme heme oxygenase-1 was observed in the HSV-1-infected brain; this increased expression was strongly inhibited by l-NMMA treatment. These data suggest that the high level of NO produced by iNOS is a pathogenic factor in HSV-1-induced encephalitis in rats.

Animals↗

PubMed1999

Poly(L-lysine)-graft-dextran copolymer promotes pyrimidine motif triplex DNA formation at physiological pH. Thermodynamic and kinetic studies.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DNA↗

PubMed1999

Possible involvement of caspase-like family in maintenance of cytoskeleton integrity.

Caspases, a family of cysteine proteases, are the key effector proteins of apoptosis. These proteases cleave cellular proteins and are responsible for the destruction of the cell body during apoptosis. They are also involved in the activation of other proteins, such as cytokines. In this study, we demonstrate a novel function for these proteases. Z-Asp-CH2-DCB (Z-Asp), a general caspase inhibitor, blocked cell spreading on collagen-coated plates in a dose-dependent manner but did not affect cell viability. Caspase 3-like activity but not caspase 1-like activity was detected in adherent cells on both collagen-coated and poly-L-lysine-coated plates but not in suspended cells. The caspase 3-like activity was significantly inhibited by Z-Asp. However, only Z-Asp, not specific caspase inhibitors (Z-DEVD for caspase 3, Z-YVAD for caspase 1), was effective in the suppression of cell spreading. The inhibitory effect of Z-Asp was blocked by a phosphokinase C activator, PMA, and a Rho activator, lysophosphatidic acid (LPA), while neither a Rac activator, bradykinin, nor a Cdc42 activator, sphingosine-1 -phosphate, was effective. Immunoprecipitation demonstrated that Z-Asp downregulated the expression of focal adhesion kinase (FAK) protein, downstream of Rho signaling, in adherent cells. Our results suggest that not caspase 1 or 3 but another yet unknown caspase(s) plays an important role in the maintenance of cytoskeleton integrity via FAK protein expression, implying a new function for caspases.

3T3 Cells↗

PubMed1999

Expression of vascular endothelial growth factor promotes colonization, vascularization, and growth of transplanted hepatic tissues in the mouse.

A complex vascular network forms an important component of the liver architecture. This network is essential for the supply of oxygen and nutrients to cells and delivery of molecules for metabolic exchange. In this study, we attempted to construct a vascular network in transplanted hepatic tissues and examined the effect of such network on tissue formation. Primary hepatocytes of adult mice were transfected with vascular endothelial growth factor (VEGF) gene in vitro then transplanted with collagen beads intraperitoneally in mice. VEGF-transfected hepatocytes secreted sufficient protein of the transgene in vitro to induce proliferation of endothelial cells. In vivo, VEGF-transfected hepatocytes formed a large number of colonies and developed a significant vascular network in established tissues compared with control tissues. In addition, hepatocytes of VEGF-transfected, established tissues proliferated and formed a substantial parenchymal region. These hepatocytes were also functional as confirmed by the production of albumin. Our results suggested that VEGF expression conferred not only the formation of a vascular network but also promoted tissue formation. Our study showed that ex vivo gene transfection into hepatocytes is a useful method for the induction of liver reconstitution in vivo.

Albumins↗

PubMed1999

Effects of qing fei tang (TJ-90) on aspiration pneumonia in mice.

The effects of Qing Fei Tang (Sei-hai To in Japanese), a Chinese traditional medical mixture, on aspiration pneumonia were studied using mice inoculated with both Streptococcus pneumoniae and gastric juice as aspiration pneumoniae models. Daily (4 weeks) oral usage of Qing Fei Tang before inoculation reduced remarkably the mortality rate of mice. In this aspiration pneumonia model, xanthine oxidase (XO) activity in the lung tissues was elevated, but this elevation was remarkably decreased by use of Qing Fei Tang. These results suggest that Qing Fei Tang pretreatment can reduce oxygen radical production in inflammed lungs and may reduce the mortality for aspiration pneumonia.

Animals↗

PubMed1999

Induction of haem oxygenase-1 nitric oxide and ischaemia in experimental solid tumours and implications for tumour growth.

Induction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either Nomega-nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO+) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6-20 mg kg(-1)). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity.

Animals↗

PubMed1999

Partially degradable film/fabric composites: textile scaffolds for liver cell culture.

In this study, a composite scaffold combining textile superstructures and biomimetic glycopolymers is introduced, which may allow engineering of organotypic liver tissue in vitro. Woven poly(ethylene therephtalat) (PET) fabrics were coated on one side with a thin biodegradable polymer film (poly[D-L-lactic-co-glycolic acid] PLGA), in order to obtain a polar structure. The composite structure ensured the stability of the membrane during in vitro degradation, independently of mesh size. Matrix porosity increased when a polymer blend matrix was used. For hepatocyte culturing studies, the scaffolds were additionally coated with an artificial glycopolymer (poly[N-p-vinylbenzyl-D-lactoamide], PVLA) in order to improve cell attachment. It was observed that formation of aggregates depends on the scaffold geometry as well as on the pretreatment and medium conditions. After 4 days in culture, the pores of the fabric were filled with aggregates illustrating the possibility of immobilizing hepatocyte aggregates in well-defined spatial configurations on textile structures.

Biocompatible Materials↗

PubMed1999

Role of peroxynitrite in airway microvascular hyperpermeability during late allergic phase in guinea pigs.

We investigated the role of peroxynitrite, which is formed by a rapid reaction between nitric oxide (NO) and superoxide anion (O(2)(-)), in the airway microvascular hyperpermeability during the late allergic response (LAR) in sensitized guinea pigs in vivo. The occurrence of LAR was assessed as a 100% increase in the transpulmonary pressure, which was monitored by the esophageal catheter technique. Airway microvascular permeability was assessed by Monastral blue dye trapping between the endothelium using an image analyzer. In the LAR phase (4 to 6 h after antigen inhalation), microvascular hyperpermeability and eosinophil infiltration within the airway wall were observed. NO production and xanthine oxidase (XO)/xanthine dehydrogenase activity, which are responsible for O(2)(-) production, were enhanced during the LAR. Peroxynitrite formation assessed by nitrotyrosine immunostaining was also exaggerated at that time. The microvascular hyperpermeability during the LAR was largely reduced by NO synthase inhibitor (L-NAME, 72.7% inhibition; p < 0.05), XO inhibitor (AHPP, 60.8% inhibition; p < 0. 05) and peroxynitrite scavenger (ebselen, 81.0% inhibition; p < 0. 05). L-NAME had a small but significant inhibitory effect on airway eosinophil accumulation, but AHPP and ebselen had no effect. These results suggest that excessive production of O(2)(-) and NO occurs in the LAR. These two molecules appear to cause airway microvascular hyperpermeability via peroxynitrite formation.

Animals↗

PubMed1998

The possible effect of nitric oxide on relaxation and noradrenaline release in the isolated rabbit urethra.

We evaluated the effects of N(omega)-nitro-L-arginine (L-NNA, a nitric oxide (NO) synthase inhibitor) and carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, a NO scavenger) on NO-mediated relaxation and noradrenaline release from adrenergic nerve endings induced by electrical field stimulation in the rabbit urethra. Electrical field stimulation caused frequency-dependent relaxation of rabbit urethral smooth muscles precontracted with phenylephrine. The relaxation responses were significantly inhibited by treatment with L-NNA or carboxy-PTIO. The inhibitory effect of carboxy-PTIO was significantly weaker than that of L-NNA. Electrical field stimulation caused significant noradrenaline release from adrenergic nerve endings in the rabbit urethra. Treatment with carboxy PTIO enhanced electrical field stimulation-induced noradrenaline release, and simultaneous application of L-NNA and carboxy-PTIO did not further enhance noradrenaline release in the rabbit urethra. As carboxy-PTIO reacts only with the free radical NO, the present results suggest that free radical NO and NO-containing compounds are involved in the L-NNA-sensitive nitrergic nerve-mediated relaxation in the rabbit urethra. At the same time free radical NO has a prejunctional action by which it may inhibit noradrenaline release from adrenergic nerves.

Animals↗

PubMed1998

Poly(L-lysine)-graft-dextran copolymer: amazing effects on triplex stabilization under physiological pH and ionic conditions (in vitro).

Triplex DNA formation involving unmodified triplex-forming oligonucleotides (TFOs) is very unstable under physiological conditions. Here, we report a novel strategy to stabilize both purine and pyrimidine motif triplex DNA within the rat alpha1 (I) collagen gene promoter under physiologically relevant conditions by a poly(L-lysine)- graft -dextran copolymer. Using an in vitro electrophoretic mobility shift assay, we show that the copolymer almost completely abrogates the inhibitory effects of physiological concentrations of monovalent cations, particularly potassium ion (K+), on purine motif triplex formation involving very low concentrations of an unmodified guanine-rich TFO. Of importance, pH dependency in pyrimidine motif triplex formation involving an unmodified cytosine-rich TFO is also significantly overcome by the copolymer. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of other oligocations, like spermine and spermidine. We suggest that the ability of the graft copolymer to stabilize triplex DNA under physiologically relevant pH and salt concentrations will be a cue for further progress in the antigene strategy.

Cations, Monovalent↗

PubMed1998

A new non-viral DNA delivery vector: the terplex system.

A new DNA delivery vector (the terplex system) based on a balanced hydrophobicity and net surface charge between stearyl-poly(L-lysine), low density lipoprotein (LDL), and genetic material (i.e. plasmid DNA or antisense oligonucleotide) was developed. The pSV-beta-gal plasmid in terplex system showed a 2-5-fold increase in beta-galactosidase expression on murine smooth muscle cells (A7R5) compared to Lipofectin. Delivery of unmodified c-myb antisense oligonucleotide to A7R5 cells was also facilitated significantly by the terplex system, requiring as little as 5.4 nM of antisense oligonucleotide to achieve a 50% antiproliferative effect. Similar antiproliferative effect was observed when the c-myb antisense/terplex formulation was tested on CCD-32 Lu human lung fibroblasts. Characterization of the physical properties of the terplex system was performed using various techniques. Plasmid DNA was condensed by addition of stearyl-PLL and LDL, resulting in the terplex system of about 100 nm in diameter as shown by atomic force microscopy. A strong hydrophobic interaction between stearyl-poly(L-lysine) and LDL was registered by 1H-NMR spectrometry, showing a significant decrease in the epsilon-methylene signal of poly(L-lysine) backbone when stearyl-poly(L-lysine) was mixed with LDL; however, this phenomenon was not observed with unmodified poly(L-lysine). Agarose gel electrophoresis revealed that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-poly(L-lysine), indicating that the surface charge of the terplex system became more positive by addition of stearyl-poly(L-lysine). Zeta-potential measurement showed that the terplex system exerted a slightly positive charge (+2 mV) at a 1:1:1 weight ratio of plasmid DNA:LDL:stearyl-poly(L-lysine). The obtained results will be utilized in the design of more efficient and safer DNA delivery vectors for in vivo gene therapy.

Animals↗

PubMed1998

Mitogenic-factor-dependent regulation of lipopolysaccharide and cytokine mixture-mediated hepatocyte nitric oxide synthesis in vitro.

Hepatocellular mitogen (HGF and EGF) inhibited lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis and cellular injury in hepatocytes. Mitogenic inhibitors such as hydroxyurea and Wortmannin could not reverse EGF or HGF-inhibited NO production, whereas both of them showed some inhibitory effect on hepatocyte NO synthesis. Although activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) had no effect on hepatocyte NO synthesis, deletion of PKC activity by long-term treatment of hepatocytes with PMA abolished LPS/CM-induced NO production. In addition, pretreatment of hepatocytes with HGF and EGF also blocked LPS/CM-induced NO synthesis in the hepatocyte. These results suggest that proliferating signal is not directly involved in mitogen-inhibited NO synthesis in the hepatocyte, and LPS/CM-mediated NO synthesis is associated with the metabolic/redox state of hepatocytes.

Androstadienes↗

PubMed1998

Clonazepam release from core-shell type nanoparticles in vitro.

Block copolymers consisting of poly(gamma-benzyl L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene oxide) (PEO) as the hydrophilic block were synthesized and characterized. Core-shell type nanoparticles of the block copolymers (abbreviated as GE) were prepared by the diafiltration method. The particle size diameter obtained by dynamic light scattering of GE-1 (PBLG content: 60.5 mol%), GE-2 (PBLG content: 40.0 mol %), GE-3 (PBLG content: 124.4 mol %) copolymer was 309.9 +/- 160.9, 251.9 +/- 220.6 and 200.5 +/- 177.1nm, respectively. The shape of the nanoparticles by SEM or TEM was almost spherical. The critical micelle concentration of the block copolymers obtained by fluorescence spectroscopy was dependent on the chain length of hydrophobic PBLG. The micelle structure of the copolymers nanoparticle was very stable against sodium dodecyl sulfate. Clonazepam (CZ) was loaded onto the core part of the nanoparticle as the crystalline state. Release of CZ from the nanoparticles in vitro was dependent on the drug loading contents and PBLG chain length.

Anticonvulsants↗

PubMed1998

Modulation of enhanced vascular permeability in tumors by a bradykinin antagonist, a cyclooxygenase inhibitor, and a nitric oxide scavenger.

The mechanism of the enhanced vascular permeability and retention (EPR) effect seen in solid tumors was investigated with sarcoma 180 (S-180) in mice by using the bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin] (HOE 140), the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), and the cyclooxygenase (prostaglandin synthase) inhibitor indomethacin. In the S-180 solid tumor model, administration of HOE 140 (0.65 or 1.3 microg/kg/8 h, s.c.), PTIO (167 mg/kg/2 h, four times/8 h, i.p.), or indomethacin (5 or 10 mg/kg/day, three times, i.p.) significantly suppressed the EPR effect in the tumor, and the combined administration of these agents achieved a stronger inhibition of the EPR effect than did each compound alone. Indomethacin (10 mg/kg/day, three times) plus PTIO (167 mg/kg/2 h, four times) given i.p. had the greatest inhibition (70%) on the EPR effect. When HOE 140 was administered s.c. at a dose of 13 microg/kg/12 h for 2 weeks after tumor inoculation, growth of the solid tumor was also suppressed by 32%, by tumor weight. In the ascitic form of S-180, i.p. administration of HOE 140 at 13 microg/kg/12 h initiated immediately after tumor inoculation significantly suppressed formation of S-180 tumor ascites; the life span of ascitic S-180 tumor-bearing mice was prolonged at the same dose of HOE 140. The expression of inducible NO synthase mRNA and of cyclooxygenase 2 mRNA in S-180 tumor tissue was highly elevated, as evidenced by Northern blotting and reverse transcription-PCR and by Southern blot analyses. These results indicate that bradykinin, NO, and prostaglandins play an important role in enhanced vascular permeability in tumor tissue and sustain tumor growth. More importantly, bradykinin antagonists such as HOE 140 may be beneficial for the inhibition of tumor growth.

Animals↗