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Biomedical subjects

M Shimoyama

Publications and source records attributed to M Shimoyama.

At least 91 records · Page 5Linked to original sources
PubMed1997

Tonic block of the Na+ current in single atrial and ventricular guinea-pig myocytes, by a new antiarrhythmic drug, Ro 22-9194.

Ro 22-9194 reduced the Na+ current in the atrial myocytes as well as ventricular myocytes in a tonic block fashion. Ro 22-9194 had a higher affinity to the inactivated state Na+ channels (KdI = 3.3 microM in atrial myocytes, KdI = 10.3 microM in ventricular myocytes) than to those in the rested state (KdR = 91 microM in atrial myocytes, KdR = 180 microM in ventricular myocytes), which indicated that Ro 22-9194 had a higher affinity to the Na+ channels in atrial myocytes than in ventricular myocytes. Ro 22-9194 shifted the inactivation curve in the hyperpolarized direction in both atrial and ventricular myocytes. These findings suggest that Ro 22-9194 more strongly inhibited the Na+ channel of the atrial myocytes of the diseased hearts with the depolarized membranes potentials than the Na+ channels in ventricular myocytes.

Animals↗
PubMed1997

[Introduction of anticancer agents to phase I clinical trials].

Because about half of patients with cancer could not be cured by the present standard therapy, new effective anticancer agents should be developed and introduced at the clinical level. However, there are several important and specific issues from scientific, medical, statistical, and ethical viewpoints in the design and conducting of phase I clinical trials of new anticancer agents. Clinical safety data management is critically important in phase I clinical trials. Phase I clinical trials are vastly different from phase II or III clinical trials in terms of these issues. This paper discusses issues of early anticancer drug development to be solved or improved.

Antineoplastic Agents↗
PubMed1997

Aggregation of human blood platelets by remnant like lipoprotein particles of plasma chylomicrons and very low density lipoproteins.

Remnant like lipoprotein particles (RLP) of partially catabolised human plasma chylomicrons (CM) and very low density lipoproteins (VLDL) were separated from CM and VLDL using two monoclonal antibodies, anti apo B-100 (JI-H) and anti apo A-I (H-12) coupled to Sepharose 4B gel to form an immunoaffinity column. Lipoproteins containing apo B-100 or apo E, including VLDL and LDL adsorb to (JI-H)-gel, while CM and HDL with apo A-I adsorb to (H-12)-gel. The unbound fraction (RLP) is rich in apo B-48, apo E and apo E rich apo B-100 which has not been recognized by JI-H. The RLP fraction with a total triglyceride of 12.35 +/- 6.22 mg/ml; total cholesterol, 0.32 +/- 0.08 mg/ml and total protein, 0.72 +/- 0.12 mg/ml (mean +/- S.E.M, n = 9) was added to blood from healthy persons at 2.5-200 microliters/ml and agitated gently at 37 degrees C for 40 s. Platelet aggregation was assessed by measuring the loss of single platelets. At 2.5-10 microliters, RLP induced platelet aggregation increased with the dose of RLP, but decreased at 25-200 microliters. Scanning electron microscopy revealed that within 20 s of agitation in the presence of RLP, activated platelets had appeared on the red cell membrane and within 40 s of agitation, platelet aggregates had formed on the red cells. The platelet responses were unaffected by aspirin (10 or 20 micrograms/ml) but were inhibited by cilostazol, a phosphodiesterase type III inhibitor (0.4 to 1.6 micrograms/ml). It is likely that the platelet effect of RLP is a consequence of RLP dependent red cell-platelet interaction. This is the first report of platelet aggregation induced by RLP without an added platelet agonist.

Adenosine Diphosphate↗
PubMed1997

Suppression of nuclear factor kappa B and CD18-mediated leukocyte adhesion to the corneal endothelium by dexamethasone.

PURPOSE: To demonstrate that leukocyte adhesion to cultured corneal endothelial cells is mediated by the CD18 antigen, and to determine whether dexamethasone directly suppresses adhesion by inhibiting activation of nuclear factor kappa B (NFkappaB). METHODS: Cultured bovine corneal endothelium was stimulated for 6 hours by 40 micron/ml tumor necrosis factor alpha (TNFalpha). Dexamethasone was added 1 hour before TNFalpha stimulation in the dexamethasone group. After stimulation, neutrophils separated from a healthy human volunteer were added with or without anti-CD18 antibody. The culture plate was settled for 15 minutes at 37 degrees C, and then neutrophils were activated by N-formyl-methionyl-leucyl-phenylalanine for 5 minutes. Nonadherent neutrophils were removed by sealing and inverting the culture well. The intracellular localization of NFkappaB after TNFalpha simulation was determined by confocal immunocytochemistry using an anti-p65 antibody. RESULTS: Neutrophil adhesion to cultured corneal endothelial cells increased significantly on exposure to TNFalpha (451.4+/-45.4 cells/mm2, n = 16) compared to control (156.7+/-27.3 cells/mm2, n = 16, P < 0.01). This increased adhesion was suppressed by the addition of anti-CD18 antibody (157.6+/-25.1 cells/mm2, n = 8, P < 0.01) and by pretreatment with 10(-7) M dexamethasone (207.9+/-31.5 cells/mm2, n = 10, P < 0.01). Immunocytochemistry 60 minutes after stimulation revealed that NFkappaB was located in the cytoplasm in unstimulated cells; however, the addition of TNFalpha caused NFkappaB to translocate into the nucleus. Pretreatment with dexamethasone tapered NFkappaB translocation into the nucleus. CONCLUSIONS: Leukocyte adhesion to the corneal endothelium was shown to be mediated by CD18 expressed on activated leukocytes. Pretreatment of the endothelium with dexamethasone inhibited leukocyte adhesion; this may be due in part to the suppression of NFkappaB entry into the nucleus.

Animals↗
PubMed1997

d-Methadone is antinociceptive in the rat formalin test.

The l-isomer of methadone possesses opioid activity, whereas the d-isomer is weak or inactive as an opioid. Both d- and l-methadone have been shown to bind to the N-methyl-D-aspartate (NMDA) receptor. To determine whether d-methadone has functional, in vivo NMDA receptor antagonist activity, the antinociceptive effects of d-methadone were evaluated in the rat tail-flick and formalin tests. Cumulative dose-response analysis in the tail-flick test revealed an ED50 value for intrathecal (spinal) l-methadone of 15.6 microg/rat. In contrast, spinal d-methadone produced no antinociception at a cumulative dose of 460 microg/rat. d-Methadone in a dose range from 32 to 320 microg/rat dose-dependently reduced formalin-induced flinching behavior during phase 2 but not during phase 1 of the formalin test. These antinociceptive effects of d-methadone were not blocked by a spinal dose of naloxone that effectively antagonized an antinociceptive (tail-flick test) dose of l-methadone. d-Methadone at an intrathecal dose of 250 microg shifted the ED50 value for NMDA-induced nociceptive behaviors more than 3-fold to the right, which indicates an antagonism of these NMDA receptor-mediated effects. These results indicate that d-methadone is antinociceptive as a result of its NMDA receptor antagonist activity.

Analgesics, Opioid↗
PubMed1996

Glutamic acid 207 in rodent T-cell RT6 antigens is essential for arginine-specific ADP-ribosylation.

A rat T-cell antigen RT6.1 catalyzes NAD glycohydrolysis but not ADP-ribose transfer, even though the antigen has significant amino acid identity with eucaryotic arginine-specific ADP-ribosyltransferases. Since a highly conserved Glu in the catalytic region of these transferases is substituted with Gln at position 207 in RT6.1, we replaced the Gln with Glu, Asp, or Ala, by site-directed mutagenesis. The Glu-207 mutant produced ADP-ribosylarginine during incubation with NAD and L-arginine. The Asp-207 mutant but not the Ala-207 mutant produced ADP-ribosylarginine, but at a lower rate. In contrast, these mutations affected NAD glycohydrolase activity of RT6.1 to a much lesser extent. Kinetic studies of transferase reaction revealed that kcat of the Glu-207 mutant increased compared to findings with the Asp-207 mutant. Moreover, the mouse homologue of rat RT6 lost arginine-specific ADP-ribosyltransferase activity when Glu-207 was replaced with Gln. Thus, Glu-207 in rodent T-cell RT6 antigens is essential for transfer reaction of ADP-ribose to arginine.

ADP Ribose Transferases↗
PubMed1996

Expression of Epstein-Barr virus latent infection genes and oncogenes in lymphoma cell lines derived from pyothorax-associated lymphoma.

Malignant lymphomas frequently develop in the pleural cavity of patients with long-standing pyothorax. The term pyothorax-associated lymphoma (PAL) has been proposed for this type of tumor. Most PALs are diffuse lymphomas of the B-cell type and contain Epstein-Barr virus (EBV) DNA. We have established 2 lymphoma cell lines from biopsy specimens of PAL cases, OPL-1 and OPL-2, and examined their growth characteristics and the expression of EBV latent infection genes and oncogenes. OPL-2 exhibited a more rapid growth and higher saturation density than OPL-1, and only OPL-2 exhibited colony-forming activity in soft agar. OPL-1 and -2 were positive for B-cell differentiation markers and showed clonal surface immunoglobulins. Both line contained a single predominant form of episomal EBV DNA, indicating clonal cellular proliferation of an EBV-infected progenitor cell. OPL-1 and -2 contained type B and A EBV genome, respectively. Expression of EBV nuclear antigen (EBNA)2 mRNA and protein was detected by Northern and Western blot analysis in OPL-1, but not in OPL-2. On the other hand, the expression of latent membrane protein (LMP)1 mRNA in both OPL-1 and -2 was extremely weak and detectable only by reverse transcription-polymerase chain reaction. Protein expression of LMP1 was not observed by Western blot analysis or immunocytochemistry. Both lines expressed c-myc mRNA. Only OPL-1 expressed mRNA of c-fgr, an oncogene whose expression is upregulated by EBNA2. Both OPLs expressed bcl-2 mRNA without detectable expression of LMP1 protein.

Aged↗
PubMed1996

Subthreshold UV radiation-induced peroxide formation in cultured corneal epithelial cells: the protective effects of lactoferrin.

Acute exposure to suprathreshold ultraviolet B radiation (UV-B) is known to cause photokeratitis resulting from the necrosis and shedding of corneal epithelial cells. however, the corneal effects of low dose UV-B in the environmental range is less clear. In this study, subthreshold UV-B was demonstrated to cause non-necrotic peroxide formation in cultured corneal epithelial cells, which was attenuated by the major tear protein lactoferrin. Intracellular oxidative insults and cell viability of rabbit corneal epithelial cells (RCEC) were assessed by dual-color digital microfluorography using carboxydichlorofluorescin (CDCFH) diacetate bis (acetoxymethyl) ester, a hydroperoxide-sensitive fluoroprobe, and propidium iodide (PI), respectively. The magnitude of UV-induced oxidative insults was calibrated by concentrations of exogenously applied H2O2 which evoke compatible levels of CDCFH oxidation. Exposure of RCEC to low-dose UV-B (2.0 mJ cm-2 at 313 nm, 10.0 mJ cm-2 total UV-B) caused intracellular oxidative changes which were equivalent to those elicited by 240 microM hydrogen peroxide under the conditions of the study. The changes were dose dependent, non-necrotic, and were partially inhibited by lactoferrin (1 mg ml-1) but not by iron-saturated lactoferrin. Pretreatment with deferoxamine (2 mM) or catalase (100 U ml-1) also attenuated the UV-induced oxidative stress. The results indicate that UV-B comparable to solar irradiation levels causes significant intracellular peroxide formation in corneal epithelial cells, and that lactoferrin in tears may have a physiological role in protecting the corneal epithelium from solar UV irradiation.

Animals↗
PubMed1996

Activation of Clostridium botulinum C3 exoenzyme-catalyzed ADP-ribosylation of RhoA by K+ in a Mg2+ -dependent manner.

The effect of KCl on ADP-ribosylation of the recombinant RhoA protein catalyzed by the Clostridium botulinum C3 enzyme was studied. When the recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was incubated with C3 and [adenylate-32P]NAD, incorporation of radioactivity into the recombinant RhoA increased in the presence of KCl. The increase in ADP-ribose incorporation into RhoA due to KCl appeared in the presence of MgCl2 and was abolished by EDTA. C3 was stabilized by KCl, but the stabilization was also seen with BSA. The KCl-induced increase in the ADP-ribosylation was observed even in the presence of BSA during the modification reaction, thus the effect of KCl was not due to the stabilization of C3. While the initial rate of the reaction was increased by KCl, maximum incorporation of ADP-ribose per GST-RhoA molecule did not increase in the presence of KCl. Kinetic analysis revealed that KCl increased Vmax but did not alter Km for either NAD or RhoA. The NAD glycohydrolase activity of C3 was also increased by KCl. These results indicate that KCl directly activates the C3 enzyme.

ADP Ribose Transferases↗
PubMed1996

Exocytosis of arginine-specific ADP-ribosyltransferase and p33 induced by A23187 and calcium or serum-opsonized zymosan in chicken polymorphonuclear leukocytes.

Exocytosis is a common phenomenon in neutrophil functions. We earlier reported the co-localization of arginine-specific ADP-ribosyltransferase [EC 2.4.2.31] and its target protein p33 (mim-1 protein) in cytoplasmic granules in chicken polymorphonuclear leukocytes (so-called heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K., and Shimoyama, M. (1991) J. Biochem. 110, 388-394]. In the present study, we obtained evidence that the transferase and p33 were released into the extracellular space by the stimulus of calcium ionophore A23187 or serum-opsonized zymosan, but scarcely by phorbol myristate acetate (PMA) or N-formyl-Met-Leu-Phe (fMLP), thereby indicating the co-localization of the transferase and p33 in the azurophilic granules, and not in specific granules. [32P]ADP-ribosylation of p33 occurred in the extracellular space, induced by the stimulus of A23187 or opsonized zymosan in the presence of [32P]NAD. Our findings are interpreted to mean that heterophil transferase and p33 may be involved in neutrophil functions during processes of inflammation.

ADP Ribose Transferases↗
PubMed1996

Ketamine attenuates and reverses morphine tolerance in rodents.

BACKGROUND: The development of tolerance complicates the use of morphine to manage persistent pain. N-methyl-D-aspartate receptor antagonists can attenuate or reverse morphine tolerance. The authors studied ketamine's ability to modulate morphine tolerance. METHOD: Tolerance was produced in mice given morphine subcutaneously and was assessed by a cumulative dose-response analysis using the tail-flick test. The ability of ketamine at 0.3, 3, or 10 mg/kg given subcutaneously before and after morphine to attenuate the development of tolerance was assessed. The ability of 10 mg/kg ketamine to reverse tolerance produced by the subcutaneous implantation of morphine pellets to mice was also assessed. Rats were made tolerant to intraspinal morphine and the effects of the coadministration of 12 micrograms intraspinal ketamine were assessed. RESULTS: Morphine given subcutaneously produced a fivefold increase in the median effective (ED50) dose of morphine, which was dose-dependently attenuated by subcutaneously administered ketamine. A tenfold increase in the morphine ED50 produced by morphine pellets was completely reversed by ketamine given subcutaneously. Intraspinal morphine produced a 46-fold increase in its ED50, which was almost completely attenuated by the coadministration of intraspinal ketamine. CONCLUSIONS: Systemically administered ketamine attenuates and reverses systemically induced morphine tolerance in mice, and intraspinal ketamine attenuates tolerance produced by intraspinal morphine in rats.

Analgesia↗
PubMed1996

[Biology and clinical relevance of multidrug resistance associated antigen].

Multidrug resistance (MDR) is a main obstacle to cancer chemotherapy. P-glycoprotein(P-gp) is the first antigen discovered to be linked to MDR, and is a energy dependent plasma membrane efflux pump, which actively transports a variety of drugs and substances out of cells. P-gp can be detected by immunological or biochemical (molecular) methods, but both methods have some limitations with respect to sensitivity and specificity. Some studies have demonstrated the adverse prognostic factor of P-gp and some others have not. The discrepancy between various studies is considered to be due to the limitation of methodology for detecting P-gp and the existence of other MDR antigens, for example multidrug resistance associated protein. The clinical roles of MDR reversing agents have not yet been confirmed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Detection of arginine-ADP-ribosylated protein using recombinant ADP-ribosylarginine hydrolase.

We made use of ADP-ribosylarginine hydrolase to detect arginine-ADP- ribosylated proteins. The hydrolase was expressed in Escherichia coli as a protein fused with glutathione S-transferase (GST). The fusion protein GST-ADP-ribosylarginine hydrolase catalyzed the hydrolysis of alpha-ADP-ribosylarginine to produce ADP-ribose and arginine. Casein ADP-ribosylated with [32P]NAD and chicken heterophil arginine-specific ADP-ribosyltransferase served as a substrate for the recombinant ADP-ribosylarginine hydrolase and the released ADP-ribose was determined. Protein ADP-ribosylated by cholera toxin could serve as substrate of the hydrolase but protein ADP-ribosylated by pertussis toxin, diphtheria toxin, or C(3) enzyme of Clostridium botulinum could not. The hydrolase did not release the radioactivity incorporated into isolated rat liver nuclei incubated with [(32)P]NAD or in bovine brain cytosol incubated with [(32)P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [(32)P]NAD was removed by ADP-ribosylarginine hydrolase treatment; hence, the specific hydrolysis of ADP-ribose-arginine bond by GST-ADP-ribosylarginine hydrolase can be used to detect the arginine-ADP-ribosylated proteins in crude preparations. Arginine--ADP-ribosylated proteins in crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.

Adenosine Diphosphate Ribose↗
PubMed1995

A newly identified GPI-anchored arginine-specific ADP-ribosyltransferase activity in chicken spleen.

Arginine-specific ADP-ribosyltransferase activity was detected in chicken spleen membrane fraction and the activity was extracted by phosphatidylinositol-specific phospholipase C but not by 1 M NaCl or 1% Triton X-100. The transferase activity extracted from the spleen membrane was thiol-independent and was not inhibited by 200 mM NaCl. Zymographic analysis of the transferase, under non-reducing conditions, showed two forms of active bands corresponding to a molecular mass of 46 and 42 kDa. Thus, the presence of this novel arginine-specific ADP-ribosyltransferase, anchored to the membrane through glycosylphosphatidylinositol and different from previously cloned chicken transferases, AT1 and AT2, is being given further attention.

Animals↗
PubMed1995

Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation.

The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.

Adenosine Diphosphate Ribose↗