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Biomedical subjects

H Morimoto

Publications and source records attributed to H Morimoto.

At least 19 recordsLinked to original sources
PubMed2004

Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells.

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.

Carcinoma, Squamous Cell↗
PubMed2002

Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid.

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level.

Carcinogens↗
PubMed2001

Potent and selective ET-A antagonists. 2. Discovery and evaluation of potent and water soluble N-(6-(2-(aryloxy)ethoxy)-4-pyrimidinyl)sulfonamide derivatives.

In the preceding article,(1) we outlined the discovery and structure-activity relationship of a potent and selective ET(A) receptor antagonist 1 and its related compounds. Metabolites of 1 having potent selective ET(A) receptor antagonist activity were identified. This study suggested the metabolic pathways of 1 were considerably affected by species. Consequently, structural modification of 1 intended to improve the complexity of the metabolic pathway, and water solubility was performed. The subsequent introduction of a hydroxyl group into the tert-butyl moiety of 1 led to the discovery of our new clinical candidate, 6b, which showed a higher water solubility, a uniform metabolic pathway among species, and very high affinity and selectivity for the human ET(A) receptor (K(i) for ET(A) receptor: 0.015 +/- 0.004 nM; for ET(B) receptor: 41 +/- 21 nM).

Animals↗
PubMed2001

Potent and selective ET-A antagonists. 1. Syntheses and structure-activity relationships of N-(6-(2-(aryloxy)ethoxy)-4-pyrimidinyl)sulfonamide derivatives.

Modifications to the ET(A/B) mixed type compounds 1 (Ro. 46-2005) and 2 (bosentan) were performed. Introduction of a pyrimidine group into 1 resulted in a dramatic increase in affinity for the ET(A) receptor, and the subsequent optimization of substituents on the pyrimidine ring led us to the discovery of N-(6-(2-((5-bromo-2-pyrimidinyl)oxy)ethoxy)-5-(4-methylphenyl)-4-pyrimidinyl)-4-tert-butylbenzenesulfonamide (7k), which showed an extremely high affinity for the human cloned ET(A) receptor (K(i) = 0.0042 +/- 0.0038 nM) and an ET(A/B) receptor selectivity up to 29 000 (K(i) = 130 +/- 50 nM for the human cloned ET(B) receptor). The compound was designed on the hypothesis that the hydrogen atom of the hydroxyl group in 1 and 2 played a role not as a proton donor but as an acceptor in the possible hydrogen bonding with Tyr129. Since the incorporation of a pyrimidinyl group into the hydroxyethoxy side chain of the nonselective antagonist (1) dramatically enhanced both the ET(A) receptor affinity and selectivity, and since similar results were obtained from the benzene analogues, we put forward the hypothesis that a "pyrimidine binding pocket" might exist in the ET(A) receptor.

Animals↗
PubMed2001

An antimicrotubule agent, TZT-1027, does not induce neuropathologic alterations which are detected after administration of vincristine or paclitaxel in animal models.

One of the major dose-limiting toxicities induced by antimicrotubule antitumor agents such as vinca alkaloids and taxanes is peripheral neuropathy. The neurotoxicity of TZT-1027 (a dolastatin 10 derivative antimicrotubule agent) was thus assessed using the animal models for antimicrotubule agent-induced neurotoxicity. Rabbits were intravenously given TZT-1027 or vincristine weekly for 5 weeks. In the mouse study, TZT-1027, vincristine or paclitaxel was intravenously given every 2 days and/or weekly. Despite the neuropathologic evidence such as myelinated axonal and fiber degeneration in the peripheral nerves and in the sensory tracts of the spinal cord following the treatment with vincristine or paclitaxel, no drug-induced alteration was observed in the TZT-1027 groups. Although there are reports that some other dolastatin derivatives with antimicrotubule activity showed no neurotoxic potential in humans, the present study represents the first demonstration in experimental animals that a dolastatin derivative has no, or at least a lower, neurotoxic potential compared to other antimicrotubule agents.

Animals↗
PubMed2001

Synthesis and structure-activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors.

A series of 1-phenylpyrazoles was evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds prepared, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700) had the most potent enzyme inhibition and displayed longer-lasting hypouricemic action than did allopurinol in a rat model of hyperuricemia induced by the uricase inhibitor potassium oxonate.

Allopurinol↗
PubMed2001

The functional similarity and structural diversity of human and cartilaginous fish hemoglobins.

Although many descriptions of adaptive molecular evolution of vertebrate hemoglobins (Hb) can be found in physiological text books, they are based mainly on changes of the primary structure and place more emphasis on conservation than alterations at the functional site. Sequence analysis alone, however, does not reveal much about the evolution of new functions in proteins. It was found recently that there are many functionally important structural differences between human and a ray (Dasyatis akajei) Hb even where sequence is conserved between the two. We have solved the structures of the deoxy and CO forms of a second cartilaginous fish (a shark, Mustelus griseus) Hb, and compared it with structures of human Hb, two bony fish Hbs and the ray Hb in order to understand more about how vertebrate Hbs have functionally evolved by the selection of random amino acid substitutions. The sequence identity of cartilaginous fish Hb and human Hb is a little less than 40 %, with many functionally important amino acid replacements. Wider substitutions than usually considered as neutral have been accepted in the course of molecular evolution of Hb. As with the ray Hb, the shark Hb shows functionally important structural differences from human Hb that involve amino acid substitutions and shifts of preserved amino acid residues induced by substitutions in other parts of the molecule. Most importantly, beta E11Val in deoxy human Hb, which overlaps the ligand binding site and is considered to play a key role in controlling the oxygen affinity, moves away about 1 A in both the shark and ray Hbs. Thus adaptive molecular evolution is feasible as a result of both functionally significant mutations and deviations of preserved amino acid residues induced by other amino acid substitutions.

Amino Acid Sequence↗
PubMed2001

The skeletal muscle ryanodine receptor identified as a molecular target of [3H]azidodantrolene by photoaffinity labeling.

Dantrolene is a skeletal muscle relaxant which acts by inhibiting intracellular Ca(2+) release from sarcoplasmic reticulum (SR). It is used primarily in the treatment of malignant hyperthermia (MH), a pharmacogenetic sensitivity to volatile anesthetics resulting in massive intracellular Ca(2+) release. Determination of the site and mechanism of action of dantrolene should contribute to the understanding of the regulation of intracellular Ca(2+) release in skeletal muscle. Photoaffinity labeling of porcine SR with [(3)H]azidodantrolene, a photoactivatable analogue of dantrolene, has identified a 160 kDa SR protein with immunologic cross-reactivity to skeletal muscle ryanodine receptor (RyR) as a possible target [Palnitkar et al. (1999) J. Med. Chem. 42, 1872-1880]. Here we demonstrate specific, AMP-PCP-enhanced, [(3)H]azidodantrolene photolabeling of both the RyR monomer and a 160 or 172 kDa protein in porcine and rabbit SR, respectively. The 160/172 kDa protein is shown to be the NH(2)-terminus of the RyR cleaved from the monomer by an endogenous protease activity consistent with that of n-calpain. MALDI-mass spectrometric analysis of the porcine 160 kDa protein identifies it as the 1400 amino acid NH(2)-terminal fragment of the skeletal muscle RyR reportedly generated by n-calpain [Shevchenko et al. (1998) J. Membr. Biol. 161, 33-34]. Immunoprecipitation of solubilized, [(3)H]azidodantrolene-photolabeled SR protein reveals that the cleaved 160/172 kDa protein remains associated with the C-terminal, 410 kDa portion of the RyR. [(3)H]Dantrolene binding to both the intact and the n-calpain-cleaved channel RyR is similarly enhanced by AMP-PCP. n-Calpain cleavage of the RyR does not affect [(3)H]dantrolene binding in the presence of AMP-PCP, but depresses drug binding in the absence of nucleotide. These results demonstrate that the NH(2)-terminus of the RyR is a molecular target for dantrolene, and suggest a regulatory role for both n-calpain activity and ATP in the interaction of dantrolene with the RyR in vivo.

Adenosine Triphosphate↗
PubMed2001

Design, syntheses, and structure-activity relationships of indan derivatives as endothelin antagonists; new lead generation of non-peptidic antagonist from peptidic leads.

A new lead generation of non-peptidic ET(A) antagonists from two peptidic ET(A)-selective ones, BQ-123 and FR139317, was performed. Using computer assisted molecular modeling, a putative pharmacophore was constructed from the superposition of the reported three-dimensional structure of the cyclic peptide BQ-123 and a presumable beta-turn active conformation of the linear peptide FR139317 formed by an intramolecular hydrogen bond. According to this model, a new series of indan derivatives were designed and synthesized. Among these, 5-isobutyrylamino-6-(1-naphthylmethyloxy)-3-(2-thienyl)-1-indancarboxylic acid (1b) showed a moderate ET(A) antagonistic activity (IC50 = 28 microM).

Antihypertensive Agents↗
PubMed2001

Peplomycin-induced apoptosis in oral squamous carcinoma cells depends on bleomycin sensitivity.

Oral squamous carcinoma cell line SSCKN cells were shown to be highly sensitive to bleomycin, whereas SCCTF cells were minimally sensitive to this reagent. To determine whether the anticancer drug resistance to oral squamous carcinoma cells could be related to the degree of the drug-induced apoptosis, we examined the effects of peplomycin on induction of apoptosis in these cells. After reaching subconfluence, SCCKN and SCCTF cells were exposed to various concentrations of peplomycin. Peplomycin caused cytotoxicity in both SCCKN and SCCTF cells in a dose-dependent fashion with the maximal effect at concentrations of 1 and 10 microM, respectively, as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, we observed marked nuclear condensation and fragmentation of chromatin in SCCKN cells treated with 1 microM peplomycin. However, SCCTF cells treated with 1 microM peplomycin showed neither nuclear condensation nor fragmentation. DNA ladder formation was also detected in both cell lines by treatment with peplomycin. The induced DNA ladder formation in SCCKN and SCCTF cells was dose-dependent, with the maximal effect at concentrations of 5 and 50 microM, respectively. Bleomycin also induced DNA ladder formation in SCCKN and SCCTF cells with different sensitivities. Mitomycin C induced DNA laddering in both SCCKN and SCCTF cells; however, the intensity of DNA ladder formation was almost the same in both cell lines. The present results indicate that peplomycin-induced apoptosis in oral squamous carcinoma cell lines depends on the sensitivity of these cells to bleomycin.

Antibiotics, Antineoplastic↗
PubMed2001

Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cells.

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis.

Antigens↗
PubMed2001

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.

Animals↗
PubMed2001

Clinical management of intraductal papillary mucinous tumors of the pancreas based on imaging findings.

The aim of this study was to assess the imaging findings of pathologically proven intraductal papillary-mucinous tumors of the pancreas and the natural history of follow-up cases, and to optimize the therapeutic management of patients with these tumors according to their imaging findings. All nine patients with main duct type tumors were histologically diagnosed as having adenocarcinoma or adenoma, with no hyperplastic lesion. The images failed to discriminate between the two histologic types. In 26 patients with branch duct type tumors, all but one with intraductal mural nodules or tumors of > or = 30 mm had adenocarcinoma or adenoma, regardless of the caliber of the main duct. Of the nine patients with tumors < 30 mm and no mural nodules. three had adenoma, and six had hyperplasia. All of four patients had hyperplasia, with the additional caliber of the main duct being < 6 mm. In a series of 23 cases in which the patient was followed-up, no apparent progression was found in 17 patients who had no mural nodules and tumors of < 30 mm. Given these results, patients with main duct type tumors, and those with branch duct type tumors showing mural nodules or a tumor diameter of > or = 30 mm, are at high risk of developing neoplasms, including adenocarcinoma, for which surgical resection should be considered, whereas those patients with tumors < 30 mm and no mural nodules can be followed.

Adenocarcinoma↗
PubMed2001

Mechanism of a nitric oxide donor NOR 1-induced relaxation in longitudinal muscle of rat proximal colon.

We previously suggested that nitric oxide (NO)-mediated relaxation of the rat proximal colon is not associated with change in cyclic GMP content. We further studied the intracellular mechanism of NO-induced relaxation by measuring changes in tension and intracellular Ca2+ concentration ([Ca2+]i), simultaneously. NOR 1, NO donor, relaxed the longitudinal muscle of the rat proximal colon, which was precontracted by carbachol, with a concomitant decrease in [Ca2+]. ODQ, an inhibitor of soluble guanylate cyclase, partially inhibited the relaxant effect of only higher concentrations of NOR 1, but Rp-8-Br-cGMPS, an inhibitor of cyclic GMP-dependent protein kinase (PKG), did not have any effects on the relaxant effect of NOR 1. When the preparations were transferred to normal solution after the treatment with thapsigargin, an inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, in the absence of Ca2+, contraction with a concomitant increase in [Ca2+]i occurred. NOR 1 did not show significant effects on the tension and [Ca2+]i in thapsigargin-treated preparations. In high K+-precontracted preparations, NOR 1 relaxed the preparations with a slight change in [Ca2+]i. The relaxant effect was significantly inhibited by ODQ and Rp-8-Br-cGMPS. These results suggest that NO induces the relaxation preferentially by acting thapsigargin-sensitive function of SR and in turn decreasing [Ca2+]i, although a cyclic GMP-PKG pathway is suggested under the experimental conditions of a high K+ concentration.

Animals↗
PubMed2001

Breast cancer presenting with the syndrome of inappropriate secretion of antidiuretic hormone after simple mastectomy.

A 71-year-old woman showed disorientation 7 days after simple mastectomy for right breast cancer. Computed tomography of the brain was normal. The level of serum sodium was very low (110 mEq/l), while the urine sodium level was normal. The osmolality of urine was higher (342 mosmol/kg) than that of serum (220 mosmol/kg). These data suggested a syndrome of inappropriate secretion of antidiuretic hormone. A fluid restriction, infusion of hypertonic saline and administration of diuretics gradually increased the level of serum sodium. Subsequently, disorientation disappeared. This is a rare case of the syndrome of inappropriate secretion of antidiuretic hormone caused by simple mastectomy, a relatively minor surgical procedure.

Aged↗
PubMed2001

[A case of chronic bromvalerylurea intoxication due to habitual use of commercially available nonsteroidal anti-inflammatory drugs presenting an indefinite hyperchloremia].

A 75-year-old man was admitted to our hospital with disorientation and progression of speech disturbance and gait disturbance. He had been given a diagnosis of cervical spondylosis about four years previously, and gait disturbance and numbness in his extremities have been gradually increasing. Hyperchloremia and a careful history taking, which led to the discovery of habitual use of an analgesic containing bromvalerylurea, suggested bromism. A high level of bromide in serum yielded a diagnosis of bromism. Disorientation and speech disturbance were treated and improved by infusion diuresis. Gait disturbance only partly improved. There is a possibility that not only cervical spondylosis, but also chronic bromvalerylurea intoxication, may have contributed to the neurological disturbance resulting in gait disturbance and numbness. Bromvalerylurea, which is contained in many commercially available analgesics, should be noted as a possible cause of neurological disturbance.

Aged↗
PubMed2000

Design and synthesis of a transferable farnesyl pyrophosphate analogue to Ras by protein farnesyltransferase.

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its membrane localization and is required for both its biological activity and ability to induce malignant transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue, 8-anilinogeranyl pyrophosphate 3 (AGPP), in which the omega-terminal isoprene unit of the farnesyl group has been replaced with an aniline functionality. The key steps in the synthesis are the reductive amination of the alpha,beta-unsaturated aldehyde 5 to form the lipid analogue 6, and the subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph(3)PCl(2) followed by displacement with [(n-Bu)(4)N](3)HP(2)O(7) to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase) and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase type I (GGTase I) (K(i) = 0.06 microM, IC(50) = 20 microM) or squalene synthase (IC(50) = 1000 microM). AGPP is the first efficiently transferable analogue of FPP to be modified at the omega-terminus that provides a platform from which additional analogues can be made to probe the biological function of protein farnesylation. AGPP is the first example of a class of compounds that are alternate substrates for protein isoprenylation that are not inhibitors of squalene synthase.

Alkyl and Aryl Transferases↗