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Biomedical subjects

M Ohno

Publications and source records attributed to M Ohno.

At least 253 records · Page 14Linked to original sources
PubMed1995

[Greenfield filter placement following successful cardiopulmonary resuscitation in a patient with sudden death due to pulmonary embolization].

A 54-year-old man was admitted as an emergency following an episode of syncope and severe dyspnea. The clinical presentation and laboratory data strongly suggested pulmonary embolism, so intravenous thrombolytic therapy was performed. Immediately following the initiation of thrombolytic therapy, the patient experienced cardiopulmonary arrest, but he was successfully resuscitated. Ascending venography of the left lower limb demonstrated a large intraluminal thrombus which was apparently floating between the femoral vein and inferior vena cava. In addition to the anticoagulation therapy, a Greenfield filter was implaced intravenously to prevent recurrent embolization.

Cardiopulmonary Resuscitation↗
PubMed1995

Human thrombin receptors are insensitive to thrombin-like snake venom enzymes.

Thrombin-like snake venoms enzymes, flavoxobin, and okinaxobin I isolated from Trimeresurus flavoviridis and Trimeresurus okinavensis, respectively, were examined in SH-EP cells and evaluated whether or not they can activate human thrombin receptors. Flavoxobin was almost completely inactive in both assays for phosphoinositide turnover and DNA synthesis. In contrast, okinaxobin I stimulated phosphoinositide turnover in a dose dependent manner, but considerably weakly. The EC50 value was about 100 nM, which was 4,000 times larger than that of alpha-thrombin. This stimulation was not inhibited by hirudin, an effective inhibitor of alpha-thrombin. Okinaxobin I also induced a very weak stimulation of DNA synthesis. These results suggest that thrombin-like snake venom enzymes interact with human thrombin receptors in inefficient ways. Weak interactions of the enzymes with thrombin receptor and inhibitor were ascribed to the incomplete formation of a lysine-cation cluster necessary for electrostatic molecular recognition.

Amino Acid Sequence↗
PubMed1995

Aspects of splice site selection in constitutive and alternative pre-mRNA splicing.

RNA splicing is an indispensable step for expression of many eukaryotic genes. Combinations of 5' and 3' splice sites should be correctly selected in both constitutive and alternative splicing. Recent studies have revealed mechanisms of alternative splicing in some systems, in which specific regulators play vital roles in splice site selection. On the other hand, essential splicing factors such as SR proteins modulate splice site usage of general machinery. Specific regulators and splicing factors such as SR proteins have some common structural features. With these related components, a similar machinery of splice site selection is involved in constitutive and alternative splicing.

Alternative Splicing↗
PubMed1994

Somatostatin alleviates impairment of working memory induced by hippocampal muscarinic M1 receptor blockade in rats.

The effects of somatostatin on the impairment of working memory induced in rats by blockade of hippocampal muscarinic M1 or NMDA receptors were examined, using a three-panel runway task. Both the muscarinic M1 receptor antagonist pirenzepine (1.0 microgram/side) and the competitive NMDA receptor antagonist CPP ((+/-)-3(2-carboxypiperazin-4-yl)-propyl-1-phosphonoic acid) (32 ng/side) significantly increased the number of working memory errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points), when injected bilaterally into the dorsal hippocampus. This effect of intrahippocampal pirenzepine on working memory was alleviated by concurrent injection of 0.32 microgram/side somatostatin. However, concurrent somatostatin (0.1 or 0.32 microgram/side) had no significant effect on the intrahippocampal CPP-induced increase in working memory errors. These results suggest that somatostatin ameliorates the impairment of working memory resulting from hippocampal muscarinic M1 receptor blockade, possibly through activation of cholinergic functions.

Animals↗
PubMed1994

NMDA receptor-mediated expression of Fos protein in the rat striatum following methamphetamine administration: relation to behavioral sensitization.

In order to clarify the possible involvement of N-methyl-D-aspartate (NMDA) receptors in mediating striatal Fos protein induction and behavioral sensitization after methamphetamine administration, we examined the effects of non-competitive NMDA receptor antagonist MK-801 on these phenomena in rats. A single administration of 1.0 and 5.0 mg/kg methamphetamine resulted in a dose-dependent increase in Fos-immunoreactive cells in the medial striatum. Prior exposure to 5.0 mg/kg methamphetamine enhanced ipsilateral rotational behavior in response to subsequent methamphetamine administration in unilateral nigral-lesioned rats (sensitization). Pretreatment with 1.0 mg/kg MK-801 completely prevented both the expression of striatal Fos protein and the development of acute behavioral sensitization following a single injection of 5.0 mg/kg methamphetamine. These results suggest that NMDA receptor-mediated mechanisms contribute to the expression of striatal Fos protein associated with behavioral sensitization that follows exposure to methamphetamine.

Animals↗
PubMed1994

Cloning of a complementary DNA encoding an 80 kilodalton nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region.

Amino Acid Sequence↗
PubMed1994

Differential roles of two consecutive phenylalanine residues in thrombin receptor-tethered ligand peptides (SFFLRNP) in thrombin receptor activation.

A synthetic heptapeptide H-Ser-Phe-Phe-Leu-Arg-Asn-Pro-NH2, which corresponds to the ligand peptide latent in rodent thrombin receptors, was able to activate the thrombin receptor with no thrombin. In order to evaluate the structural requisites of two consecutive phenylalanines, three sets of analogs with substitutions at position either 2 or 3 were synthesized and examined for their stimulatory activity in phosphoinositide turnover in SH-EP epithelial-like cells. The replacement of Phe-2 by Ala completely eliminated the activity, while that of Phe-3 retained about 50% activity with a full stimulation. The Phe/Leu substitution resulted in a large increase (37-fold) in EC50 value for Phe-2, but in insignificant change for Phe-3. Substitution of para-fluorophenylalanine ((p-F)Phe) for Phe-2 enhanced strongly (4-fold) the activity, in contrast to a reduction by the Phe-3/(p-F)Phe substitution. Elimination of either Phe-2 or Phe-3 resulted in a complete loss of activity. These results indicated that Phe-2 and Phe-3 play different roles in the receptor activation. A highly specific aromatic phi-phi interaction was suggested between Phe-2-phenyl and thrombin receptor binding site, while Phe-3 appeared to be important for retaining a bioactive conformation.

Amino Acid Sequence↗
PubMed1994

Blockade of hippocampal M1 muscarinic receptors impairs working memory performance of rats.

In order to clarify the roles of hippocampal M1 and M2 muscarinic receptors in working and reference memory performance of rats, the effects of intrahippocampal injections of selective antagonists at both receptors on this behavior were examined with a three-panel runway task. In the working memory task, the M1 muscarinic receptor antagonist pirenzepine, injected bilaterally at 0.32 and 1.0 microgram/side into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). This effect of intrahippocampal pirenzepine (1.0 microgram/side) on working memory was attenuated by concurrent injection of 10 micrograms/side AF102B, the selective M1 muscarinic receptor agonist. Intrahippocampal injection of the M2 muscarinic receptor antagonist methoctramine at doses up to 1.0 microgram/side had no significant effect on the number of working memory errors. Intrahippocampal methoctramine injection at 3.2 micrograms/side produced a significant increase in working memory errors, an effect that was reversed by concurrent injection of 10 micrograms/side AF102B. Concurrent injection of 0.32 microgram/side methoctramine significantly reduced the increase in working memory errors induced by intrahippocampal pirenzepine (1.0 microgram/side). In the reference memory task, neither pirenzepine nor methoctramine affected the number of errors when injected into the hippocampus at doses up to 1.0 and 3.2 micrograms/side, respectively. These results suggest that processes mediated by M1 muscarinic receptors in the hippocampus are involved in working memory, but not in reference memory, and that blockade of hippocampal M2 muscarinic receptors ameliorates working memory deficits produced by M1 muscarinic blockade, possibly by increasing acetylcholine release.

Animals↗
PubMed1994

Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.

Amino Acid Sequence↗
PubMed1994

Simultaneous determination of dihydrocodeine and its metabolites in dog plasma by high-performance liquid chromatography with electrochemical and ultraviolet detection.

An HPLC method with electrochemical and UV detection was established for the simultaneous determination of dihydrocodeine and its metabolites, dihydronorcodeine, dihydromorphine, and dihydrocodeine glucuronide, in dog plasma using N-ethylnormorphine as the internal standard. The method involved sample pretreatment with a C18-bonded disposable column, and the injected fraction was separated and detected on the C18-bonded column with serially coupled UV and coulometric detectors. Dihydromorphine was detected with the coulometric detector at 0.4 V, and dihydrocodeine and dihydronorcodeine at 0.8 V. Dihydrocodeine glucuronide was detected with UV at 210 nm. Recoveries of the studied compounds were quantitative at the individual assay ranges, and validation of the assay gave results that were satisfactory in terms of within-run or between-run precision and accuracy. Lower limits of quantitation were 2 ng/ml for dihydrocodeine and dihydronorcodeine, 0.5 ng/ml for dihydromorphine, and 200 ng/ml for dihydrocodeine glucuronide.

Animals↗
PubMed1994

Interactions of N-terminal fragments of groups I and II phospholipases A2 with phospholipid bilayers and their surface recognition properties.

In order to elucidate the roles of the N-terminal segments of groups I and II phospholipases A2 (PLA2s) which have been known to have alpha-helical structure and have been assumed to be involved in the water/lipid interface recognition site, the peptides corresponding to the N-terminal moieties of group I PLA2 (Naja naja atra) and group II PLA2s (Trimeresurus flavoviridis and Crotalus atrox) were synthesized and their interactions with model membranes were studied. Circular dichroism spectra showed that N-terminal peptides of both groups I and II PLA2s took alpha-helical structure in trifluoroethanol but no significant secondary structure in buffer (pH 8.0). In the presence of acidic liposomes, N-terminal fragments of group II PLA2s formed alpha-helical structure, while that of group I PLA2 remained unaffected. The hydrophobic moments showed that amphipathicities of N-terminal fragments of group II PLA2s are evidently larger than those of N-terminal fragments of group I PLA2s. The leakage of carboxyfluorescein from acidic liposomes was induced only with group II PLA2 peptides. Large blue shift and increase in intensity of tryptophan fluorescence were also observed for group II PLA2 peptides when interacting with acidic liposomes. Such difference in the modes of interactions with lipid bilayers between N-terminal peptides of groups I and II PLA2s appears to be due in large part to the difference in intrinsic alpha-helix forming properties of their amino acid sequences. It is inferred that N-terminal amphipathic alpha-helical structures of group I PLA2s are possibly formed by assistance of a neighboring chain bridged by Cys-11 and Cys-77.

Amino Acid Sequence↗
PubMed1994

Intrahippocampal administration of a glycine site antagonist impairs working memory performance of rats.

The effects of 7-chlorokynurenic acid, an antagonist at the glycine site associated with the NMDA receptor/channel complex, on memory functions were investigated using a three-panel runway task. In a working memory task, 7-chlorokynurenic acid at a dose of 3.2 micrograms/side, injected bilaterally into the dorsal hippocampus, significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points). The working memory deficit induced by intrahippocampal 7-chlorokynurenic acid (3.2 micrograms/side) was reversed by concurrent injection (32 micrograms/side) of D-serine, the glycine site agonist. In a reference memory task, 7-chlorokynurenic acid had no effect on the number of errors when injected into the hippocampus at doses up to 3.2 micrograms/side. These results suggest that activation of the hippocampal glycine site coupled to the NMDA receptor is required for working memory function in rats.

Animals↗
PubMed1994

Heterotypic sprouting of serotonergic forebrain fibers in the brindled mottled mutant mouse.

The brindled mottled mouse has a mutation on the X-chromosome which causes alterations in copper metabolism. One role for copper is as a cofactor for dopamine-beta-hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine (NE). This may explain the fact that the hemizygous males have low concentrations of NE, as well as high concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in the brainstem and forebrain. The present study quantified serotonin (5-HT) immunoreactive fibers in the cerebral cortex and striatum of hemizygous males and control littermates on postnatal (P) days 7, 10, 12 and 14. The density of 5-HT immunoreactive fibers was measured using a digitized imaging system in conjunction with darkfield microscopy. Measurements of 5-HT innervation showed an age-dependent increase in density of 5-HT immunoreactive fibers in all layers of the cerebral cortex, with fiber density in brindled mice approximately 70% greater than controls by P14. High performance liquid chromatography confirmed the increased concentrations of 5-HT and 5-HIAA, and the low concentration of NE, in several regions. We believe that these results are an example of heterotypic sprouting of 5-HT neurons, similar to that observed in neonatal rats given 6-hydroxydopamine (6-OHDA). If so, these data provide the first description of 5-HT heterotypic sprouting in mice, and the first description of 5-HT heterotypic sprouting resulting from a natural disease state, rather than an experimentally induced lesion.

Animals↗
PubMed1994

Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation.

It has been reported that a peptide corresponding to the S4 segment in sodium channel protein is able to form voltage-dependent cation-selective ion channels (Tosteson, M. T., Auld, D. S., and Tosteson, D. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 707-710). However, biological and other physical properties remain unexamined. In the present study, three peptides, H-(Ala-Arg-Leu)8-OH (ARL8), H-(Val-Arg-Leu)8-OH (VRL8), and H-(Leu-Arg-Leu)8-OH (LRL8) which were designed on the basis of the S4 segment and expected to form 3(10)-helix, were synthesized and examined with regard to conformational change by the interaction with membranes, membrane perturbation ability, ion channel formation, and antimicrobial activity. According to CD spectra, these peptides were found to form a 3(10)-helical structure in the presence of dipalmitoyl-DL-alpha-phosphatidylcholine/dipalmitoyl-DL-alpha- phosphatidylglycerol (3:1) liposomes. The experiment of the peptide-induced leakage of carboxyfluorescein from liposomes showed that all the peptides had a strong ability to perturb membranes. The peptides were able to form cation-selective ion channels in planar asolectin lipid bilayers. The conductances of the ion channels were small (approximately 2 picosiemens for VRL8 and LRL8 and approximately 23 picosiemens for ARL8), suggesting that the peptides produce narrow pores or wider pores with certain permeable barriers that are a portion of the whole channels. The differences in their conductances depend possibly on the sizes of the side chains of Ala, Val, and Leu residues. However, non of the peptides showed antimicrobial activity (minimum inhibitory concentrations, > 50 micrograms/ml). Here, we present the first evidence that the peptides can form 3(10)-helical structures with long chain lengths in a lipid bilayer environment.

Amino Acid Sequence↗