Biomedical subjects

M Kawase

Publications and source records attributed to M Kawase.

At least 37 records · Page 2Linked to original sources

PubMed2000

Ganglioside GD3 and its mimetics induce cytochrome c release from mitochondria.

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.

Animals↗

PubMed2000

Effect of hypotension severity on hippocampal CA1 neurons in a rat global ischemia model.

Neuronal death in the hippocampal CA1 subregion has been shown to occur in a delayed manner after transient global ischemia. The 2-vessel occlusion model is one of the most frequently used global ischemia paradigms in rodents. Although researchers often fail to induce bilateral delayed CA1 neuronal death, the importance of hypotension severity has not been fully discussed. We induced 10 min of global ischemia with 2-vessel occlusion and various severities of hypotension in rats, and the subsequent neuronal damage and neurogenesis in the hippocampal CA1 pyramidal cell layer were immunohistochemically studied. Neuronal apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL). The mean arterial blood pressure of 31-35 mmHg was the most appropriate range of hypotension in this model because of low mortality and consistent bilateral CA1 injury. Most of the neurons in the CA1 pyramidal cell layer lost neuron specific nuclear protein and became TUNEL-positive 3 days after ischemia. There was no evidence of apoptosis or neurogenesis at 7-28 days. There were ischemia-tolerant neurons in the CA1 pyramidal cell layer that survived delayed neurodegeneration, however, further studies are necessary to characterize the property of these neurons.

Animals↗

PubMed2000

The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice.

Release of mitochondrial cytochrome c into the cytosol is a critical step in apoptosis. We have reported that early release of cytochrome c in vivo occurs after permanent focal cerebral ischemia (FCI) and is mediated by the mitochondrial antioxidant manganese superoxide dismutase (SOD). However, the role of reactive oxygen species produced after ischemia-reperfusion in the mitochondrial apoptosis process is still unknown, although overexpression of copper/zinc-SOD (SOD1), a cytosolic isoenzyme, protects against ischemia-reperfusion. We now hypothesize that the overexpression of SOD1 also prevents apoptosis after FCI. To address this issue, we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and in SOD1 transgenic (Tg) mice after transient FCI. Cytosolic cytochrome c was detected as early as 2 hr after reperfusion, and correspondingly, mitochondrial cytochrome c was significantly reduced after FCI. Cytosolic cytochrome c was significantly lower in the SOD1 Tg mice compared with wild types 2 (p < 0.0001) and 4 (p < 0.05) hr after FCI. Apaf-1, which interacts with cytochrome c and activates caspases, was constitutively expressed in both groups of animals, with no alteration after FCI. Double staining with cytochrome c immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between cytosolic cytochrome c expression and DNA fragmentation. A significant amount of DNA laddering was detected 24 hr after ischemia and was reduced in SOD1 Tg mice. These data suggest that SOD1 blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after transient FCI.

Animals↗

PubMed2000

Quantitative determination of dihydroetorphine in rat plasma and brain by liquid chromatography-tandem mass spectrometry.

The extraordinarily strong analgesic dihydroetorphine (DHE) was registered as one of the most strictly controlled narcotic drugs by the United Nations in 1999. However, an effective detection method for DHE in biological samples has not yet been established. We developed a quantitative method for assay of DHE in rat plasma and brain by liquid chromatography-tandem mass spectrometry equipped with an ionspray interface. A 0.5-ml volume of plasma and brain homogenate spiked with buprenorphine (internal standard) was purified by the solid-phase extraction column Bond Elute Certify. DHE produced numerous weak fragment ions by collision induced dissociation. Therefore, collision energy was utilized to decompose the interferences, and the protonated molecular ion was used for both precursor and product ion monitoring. As a result of the method validation, the dynamic concentration range was determined as 0.05-10 ng/ml. DHE in these samples was stable for 2 months at -4 degrees C and for 24 h at ambient temperatures. Using the present method, DHE was detected in rat plasma and brain tissue after intravenous injection (0.5 microg/kg).

Analgesics, Opioid↗

PubMed2000

Structure and function of a novel coliphage-associated sialidase.

A coliphage named 63D, isolated previously, associated sialidase as a component of phage particles. In order to localize the enzyme in phage particles, phages were partially destroyed by sonication, and the disrupted particles were size fractionated using a sucrose density gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay and electron micrography of the fractions revealed the enzyme to be composed of four identical subunits with a molecular mass of 90 kDa, and the subunits were cross-linked by disulfide bonds. Electron micrographic observation indicated that six enzyme molecules were localized in a phage tail plate as a hexagonal array.

Cell Fractionation↗

PubMed2000

Heart rate variability during massive hemorrhage and progressive hemorrhagic shock in dogs.

PURPOSE: To investigate the sequential changes in heart rate (HR), autonomic nervous activity presented by the spectral analysis of heart rate variability (HRV), hemodynamics and metabolism during massive hemorrhage and progressive hemorrhagic shock in dogs. METHODS: Twelve dogs were subjected to acute massive hemorrhage until mean arterial pressure (MAP) reached 50 mm Hg. Then bleeding was stopped and they were allowed to reach a plateau phase. They were divided, post hoc, into bradycardic or tachycardic groups according to their HR response to the acute massive hemorrhage. After reaching a plateau phase, the dogs were further bled to keep their MAP around 50 mmHg (progressive hemorrhagic shock). Their heart rate power spectra were quantified into low-frequency (LF) (0.04-0.15 Hz) and high-frequency (HF) (0.15-0.4 Hz) components. RESULTS: In the bradycardic group, both LF and HF increased after massive hemorrhage, but during progressive hemorrhagic shock these components decreased while HR increased. In the tachycardic group, LF increased after massive hemorrhage, but during progressive hemorrhagic shock LF decreased with continuous suppression of HF CONCLUSION: Massive hemorrhage caused two types of HR response: bradycardia and tachycardia. The HRV profile showed differential autonomic characteristics, and could be a valuable tool in assessing various degrees of hemorrhagic shock.

Animals↗

PubMed2000

Spirostanols obtained by cyclization of pseudosaponin derivatives and comparison of anti-platelet agglutination activities of spirostanol glycosides.

Naturally occurring saponins 3 and 4 have a normal type F ring and alpha-arranged CH(3)-21 group. Treatments of pseudosaponin peracetates 18 and 19 derived from 3 and 4, respectively, with alcoholic KOH, followed by acidification with acetic acid, gave spirostanols 20 and 22 having iso type F rings as major products. Structural analyses of sapogenins and saponins derived from pseudo derivatives 11, 12, 18 and 19 were performed by comparisons of their 1H-NMR spectral data and the X-ray analytical data of 3-O-p-bromobenzoyl sarsasapogenin 7, 3-O-acetyl diosgenin 13 and saponin 20. The mechanisms of ring-closure reaction of the side chain at C-22 of pseudosapogenins and pseudosaponins were deduced using stereomodels of the spirostanols derived from 11 under various reaction conditions. Inhibitory activities of saponin diglycosides 3, 4, 20, 21 and 25 on human platelet agglutinations induced by ADP and ristocetin were compared.

Adenosine Diphosphate↗

PubMed2000

Immunomodulating effect of 2,3,4,5-tetrahydro-1H-3-benzazepines (a new class of non-nucleoside inhibitors of reverse transcriptase).

The effects of newly synthesized, reverse transcriptase inhibitors, 3-benzazepines, for their effects on natural killer (NK) cell and blast transformation in human peripheral blood mononuclear cells were investigated. The most effective reverse transcriptase inhibitors were KF1, KF2 and KF3, which primarily suppressed immunological functions. Besides the inhibition of T cell proliferation, the benzazepines also show inhibitory effect on NK cell functions, in particularly, against large granular lymphocytes and monocytes. The B lymphocytes and Fc mediated killer functions were less inhibited.

Adjuvants, Immunologic↗

PubMed2000

Chemistry and biological activity of new 3-benzazepines.

This review summarizes our experiments investigating structure-activity relationships of 3-benzazepines. Three 7, 8-dihydroxy-3-benzazepines [7-9] were cytotoxic to human promyelotic leukaemia HL-60 cells. Compound [9] showed the highest cytotoxicity and the activity was twice as high as that of dopamine (DA, [11]). Three active compounds [7-9] produced radicals, whereas other less potent benzazepines [1-6, 10] did not produce radicals. Furthermore, cytotoxic 3-benzazepines [7-9] also enhanced the decay of ascorbic acid in rat brain homogenate. Two 7,8-dimethoxy-3-benzazepines [5, 10] were able to form a complex with the replicative form of plasmid DNA. The multidrug resistance (MDR) P-glycoprotein (Pgp) efflux pump of mouse lymphoma cells was inhibited by three compounds [5, 8, 10]. Compound [8] has the highest activity in MDR reversal and is two times more potent than verapamil. Three cytotoxic 3-benzazepines [7-9] showed inhibitory effects against reverse transcriptase (RT) of Moloney leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Synthesis and biological activity of N-acylphenothiazines.

Previous studies have demonstrated the relationship between radical intensity and cytotoxic activity in water-soluble compounds. This relationship was investigated in lipophilic compounds. Several N-acylphenothiazines showed higher cytotoxic activity against human leukemic and squamous carcinoma cell lines than phenothiazine, the parent compound. Electron spin resonance (ESR) spectroscopy showed that these active compounds produced much lower amounts of radicals than phenothiazine. Several compounds failed to inhibit the cytopathic effects of human immunodeficiency virus (HIV) infection in MT-4 cells. It suggested that the radical-mediated-mechanisms has not involved in the induction of cytotoxic activity by lipophilic compounds, such as N-acylphenothiazines.

Anti-HIV Agents↗

PubMed2000

Molecular cloning and characterization of a novel bacteriophage-associated sialidase.

Bacteriophage 63D, previously isolated from sewage, is associated with alpha-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for sigma70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.

Journal Article↗

PubMed2000

Continuous production of sialyllactose from colominic acid using a membrane reactor.

Continuous production of sialyllactose, as a typical sialylsaccharide, was examined using a membrane reactor. The synthesis of sialyllactose through the transfer reaction catalyzed by neuraminidase, has been reported to be a suitable process for industrial production, but it still has drawbacks such as a low yield, high enzyme cost, and hydrolysis by the enzyme of sialyllactose formed. We attempted to solve these problems by utilizing an appropriate membrane reactor system. We first investigated the effects of various reaction conditions on sialyllactose productivity, and found that the productivity was independent of the enzyme concentration and reaction temperature but dependent on the substrate and buffer concentrations and the hydraulic retention time (HRT). We then selected a suitable membrane that allowed sialyllactose to permeate but rejected the substrate and enzyme. Finally, we constructed a membrane reactor system with a cut-off molecular weight of 3000 and applied it to continuous sialyllactose production from colominic acid at an HRT of 80 min. Using substrate concentration of 25 g/l the system performed with a high level of productivity and gave a good yield, while maintaining high transfer ratio of 4-5% over a 160-h test period.

Journal Article↗

PubMed2000

Synthesis of sulfated colominic acids and their interaction with fibroblast growth factors.

Colominic acid, an alpha (2-->8)-linked poly(sialic acid), was sulfated and characterized by NMR spectroscopy. During sulfation, the secondary hydroxyl group at C-4 had almost the same reactivity as the primary hydroxyl group at C-9, while the secondary hydroxyl group at C-7 was hardly substituted. Analysis by molecular modeling suggested that the lack of substitution at C-7 was due to a steric hindrance. A mobility shift assay indicated that FGF-2 bound to the sulfated colominic acid. Synthetic sulfated colominic acid potentiated the mitogenic activity of FGFs for fibroblasts in the same manner as heparin. Sulfated colominic acid with a low degree of sulfation was able to potentiate FGF activity. Regardless of the degree of sulfation, sulfated colominic acid-induced cytotoxicity was not observed. It was suggested that the carboxyl groups in sulfated colominic acid cooperate with the sulfate groups to reinforce the interaction with FGFs and to reduce the cytotoxicity of sulfated colominic acids.

3T3 Cells↗

PubMed2000

Distinctive distribution of human papillomavirus type 16 and type 20 DNA in the tonsillar and the skin carcinomas of a patient with epidermodysplasia verruciformis.

BACKGROUND: Epidermodysplasia verruciformis (EV) is a rare skin disease characterized by disseminated pityriasis versicolor-like or flat wart-like lesions and by the development of skin carcinomas. It is well established that specific cutaneous human papillomaviruses (EV-HPVs) are associated with both benign and malignant skin lesions in EV patients. However, little is known of the relationship between HPV and the mucosal lesions of EV patients. OBJECTIVES: To detect and identify HPV types associated with skin and mucosal lesions of an EV patient. PATIENT/METHODS: We investigated the skin carcinoma and the coexisting tonsillar carcinoma of a 41-year-old man with EV. Histopathologically, both lesions were squamous cell carcinomas. We analysed these two lesions by immunohistochemistry, in situ hybridization, and by molecular virology. RESULTS: Neither skin nor tonsillar lesions exhibited positivity for HPV capsid antigen by immunohistochemistry. By Southern blot hybridization, however, the skin carcinoma harboured 'EV-specific' HPV20 DNA, while the tonsillar carcinoma harboured 'genital' HPV16 DNA. In addition, in situ hybridization localized the respective viral DNA in the corresponding lesion. CONCLUSIONS: The results indicate that EV-HPV could be responsible for the development of the skin carcinoma, but not the mucosal carcinoma in this patient.

Adult↗

PubMed2000

Immobilization of ligand-modified polyamidoamine dendrimer for cultivation of hepatoma cells.

Cationic polyamidoamine dendrimers are known to be highly branched cascade polymers. The core part of these polymers, tris(2-aminoethyl)amine, was immobilized onto polystyrene plates to which animal cells do not adhere. using photoreactive 4-(3-trifluoromethylazirino) benzoyl-N-succinimide (TDBA-OSu). Cells of a rat hepatoma cell line, H4-II-E-C3, adhered to a surface immobilized with a first-generation dendrimer probably through interactions between the terminal amino groups of the dendrimer and the cell membranes. The adhered cells were viable, could proliferate, and exhibited urea synthetic activity. The modification of the terminal amino groups with fructose increased the final number of cells obtained after 5 days of cultivation. Multigeneration dendrimers were prepared by repeated linkage of tris(2-aminoethyl)amine with the amino groups. Theoretically, the number of terminal amino groups available for ligand modification is twice as much for each generation of dendrimer growth. Cells cultivated on multigeneration fructose-modified dendrimers exhibited enhanced urea synthetic activity. The use of ligand-modified dendrimers is, therefore, considered to be very promising for the construction of bioartificial organs based on cultivation of the animal cells.

Animals↗

PubMed2000

Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid.

Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.

Animals↗

PubMed2000

Transdermal delivery of the potent analgesic dihydroetorphine: kinetic analysis of skin permeation and analgesic effect in the hairless rat.

Dihydroetorphine is an extraordinarily strong opioid analgesic. To assess its effectiveness after topical application in hairless rats we have examined the kinetic analysis of skin permeation through excised skin and the in-vitro reservoir effect of skin, and have investigated the predictability of plasma concentration and analgesic effect following in-vivo transdermal application. Dihydroetorphine was moderately permeable from an aqueous suspension through excised hairless rat skin. Dihydroetorphine flux from drug-dispersed pressure-sensitive adhesive tape was threefold that from the applied aqueous suspension. The fluxes through the abdominal and the dorsal skin during tape application fitted the Fickian diffusion equation well after the tape was removed peeling off the outer layer of the stratum corneum. The relationship between the plasma concentration and the analgesic effect was examined for four different rates of infusion of dihydroetorphine. A non-linear pharmacokinetic disposition was observed. Following abdominal (0.28 cm2, 20 microg) and dorsal (0.50 cm2, 35 microg) applications of the dihydroetorphine tape, plasma concentration (0.2-0.8 ng mL(-1)) and analgesic effect were maintained at a suitable level, for more than 8 h, until removal of the tape. These profiles were predictable using the combined equation for percutaneous absorption, disposition and the analgesic effect, but the analgesic effect was slightly lower than the predicted value. The results show that it was possible to control the plasma concentration and the analgesic effect of dihydroetorphine by topical application of the analgesic using pressure-sensitive adhesive tape in the hairless rat. It was possible to predict the result using mathematical modelling.

Administration, Cutaneous↗

PubMed2000

The Dakin-West reaction of N-alkoxycarbonyl-N-alkyl-alpha-amino acids employing trifluoroacetic anhydride.

The Dakin-West reaction of N-alkoxycarbonyl-N-alkyl-alpha-amino acids (1a-j) with trifluoroacetic anhydride in the presence of pyridine gave alpha-amido trifluoromethyl ketones (2a-j), in which probable intermediates were mesoionic 1,3-oxazolium-5-olates (munchnones). The diastereoselective reduction of 2a-f with NaBH4 gave the threo-aminoalcohols (5a-f), which may be explained by the Felkin-Anh model. This was confirmed by converting 5a-f into trans-5-trifluoromethyl-2-oxazolidinones (6a-f) in good yields.

Acetic Anhydrides↗