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Biomedical subjects

Y Masuda

Publications and source records attributed to Y Masuda.

At least 19 recordsLinked to original sources

Neural activity of chorda tympani mechanosensitive fibers during licking behavior in rats.

The chorda tympani nerve, supplying the anterior two-thirds of the tongue, contains gustatory and mechanosensitive afferent fibers. We have analyzed discharge patterns in rats of various fibers recorded from dissected nerve filaments during licking behavior of which 4 were taste-sensitive and 12 mechanosensitive. The incidence of these two types were estimated electrophysiologically under anesthesia and their conduction velocity measured. Recordings in freely moving animals showed that the mechanosensitive fibers innervating the dorsal part of the tongue gave two burst discharges per lick, suggesting that contact of the tongue with the upper incisors and/or lip occurred during tongue protrusion and retraction. The fibers from the tip of the tongue showed one burst discharge per lick, which was the response to contact with a drinking spout. No rhythmical discharges synchronized with lick signals were observed in the fibers from the lateral part of the tongue or the taste-sensitive fibers. Such mechanoreceptor discharges were difficult to detect in recordings from the whole chorda tympani nerve. This masking of responses was due mainly to activation of a small number of mechanosensitive fibers by licking-induced mechanical stimulation. The lubricating action of saliva also decreased mechanoreceptor sensitivity. Despite their small number, the mechanosensitive fibers had axons with faster conduction velocities (larger diameter) than the taste-sensitive fibers. This was probably the reason why dissected nerve bundles more frequently showed mechanical than taste responses in conscious rats.

Action Potentials

Cloning and sequencing of a cDNA encoding a taste-modifying protein, miraculin.

A cDNA clone encoding a taste-modifying protein, miraculin (MIR), was isolated and sequenced. The encoded precursor to MIR was composed of 220 amino acid (aa) residues, including a possible signal sequence of 29 aa. Northern blot analysis showed that the mRNA encoding MIR was already expressed in fruits of Richadella dulcifica at 3 weeks after pollination and was present specifically in the pulp.

Amino Acid Sequence

Anticholinergic effects of class III antiarrhythmic drugs in guinea pig atrial cells. Different molecular mechanisms.

BACKGROUND: It is well known that vagal stimulation increases the vulnerability to atrial fibrillation via muscarinic receptor-mediated shortening of refractory period. Recently it has been reported that some class III antiarrhythmic drugs effectively terminate or prevent atrial flutter and fibrillation by prolonging atrial effective refractory period. However, effects of class III antiarrhythmic drugs on the muscarinic acetylcholine receptor-operated K+ current (IK.ACh), which is important for the repolarization phase of the action potential in atrial cells, have not been thoroughly examined. METHODS AND RESULTS: Effects of three class III antiarrhythmic drugs, d,l-sotalol, E-4031, and MS-551, on the carbachol (1 mumol/L)-induced action potential shortening and outward K+ current were examined in guinea pig atrial cells by conventional microelectrode and patch clamp techniques. In isolated left atria, d,l-sotalol (100 mumol/L), E-4031 (3 mumol/L), and MS-551 (30 mumol/L) partially reversed the carbachol-induced action potential shortening. In isolated single atrial cells, IK.ACh was activated by extracellular application of carbachol (1 mumol/L) or adenosine (10 mumol/L) or by intracellular loading of GTP gamma S (100 mumol/L). Sotalol (3 to 1000 mumol/L), E-4031 (1 to 100 mumol/L), and MS-551 (1 to 100 mumol/L) inhibited the carbachol-induced IK.ACh in a concentration-dependent manner, and their IC50 (half-maximal inhibition) values were 35.5, 7.8, and 11.4 mumol/L, respectively. However, the GTP gamma S-induced and adenosine-induced IK.ACh were inhibited by high concentrations of E-4031 and MS-551 but not by sotalol. CONCLUSIONS: Sotalol may inhibit IK.ACh by the blockade of the atrial muscarinic receptors, whereas E-4031 and MS-551 may inhibit the current not only by blocking the muscarinic receptors but also by depressing the function of the K+ channel itself and/or G proteins. These drugs may potentially be useful for the prevention and termination of atrial flutter and fibrillation through their inhibitory action on IK.ACh.

Acetylcholine

Induction of hepatic microsomal drug-metabolizing enzymes by methylsulphonyl metabolites of polychlorinated biphenyl congeners in rats.

The effect of methylsulphonyl (MeSO2) metabolites of 2,3',4',5-tetrachlorobiphenyl (tetraCB) (IU-70), 2,2',3',4',5-pentachlorobiphenyl (pentaCB) (IU-87), 2,2',4',5,5'-pentaCB (IU-101) and 2,2',3',4',5,5'-hexachlorobiphenyl (hexaCB) (IU-141), on the hepatic microsomal drug-metabolizing enzyme system was investigated in rats. The administration of 3-MeSO2-2,3',4',5-tetraCB (10 mumol/kg), 3-MeSO2-2,2',3',4',5-pentaCB (0.5 mumol/kg), 3-MeSO2-2,2',4',5,5'-pentaCB (0.5 mumol/kg) and 3-MeSO2-2,2',3',4',5,5'-hexaCB (2 mumol/kg) to rats significantly increased the contents of cytochromes P-450 and b5 and the activities of aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and benzo[a]pyrene hydroxylase. From these results, it is suggested that the 3-MeSO2 derivatives studied are possibly potent phenobarbital-like inducers of microsomal drug-metabolizing enzymes. On the other hand, 4-MeSO2-2,3',4',5-tetraCB, 4-MeSO2-2,2',3',4',5-pentaCB, 4-MeSO2-2,2',4',5,5'-pentaCB and 4-MeSO2-2,2',3',4',5,5'-hexaCB had almost no effect on both cytochrome contents and these enzyme activities. After 96 h, following administration of 2,3',4',5-tetraCB, 2,2',3',4',5-pentaCB, 2,2',4',5,5'-pentaCB and 2,2',3',4',5,5'-hexaCB (342 mumol/kg each), significant increases in contents of these two cytochromes and in activities of these enzymes were observed. The relationship between liver concentrations of 3-MeSO2-PCBs after administration of four PCB congeners and that after administration of their 3-MeSO2 derivatives, and increases in the contents of both cytochromes and activities of drug-metabolizing enzyme suggests that the 3-MeSO2 metabolites derived from PCBs studied play an important role in the induction of the drug-metabolizing enzymes by the parent PCB congeners.

7-Alkoxycoumarin O-Dealkylase

Characterization of hepatic microsomal cytochrome P-450 from rats treated with methylsulphonyl metabolites of polychlorinated biphenyl congeners.

The inducing potency of 3-methylsulphonyl(MeSO2)-2,2',4',5,5'-pentachlorobiphenyl (pentaCB), which was one of the major MeSO2 metabolites of polychlorinated biphenyls (PCBs) present in seal blubber, on the hepatic drug metabolizing enzyme activities was examined in comparison with that of the parent compound and phenobarbital (PB). The inducing fashion of the above enzymes and changes in the contents of PB-inducible P-450 forms by 2,3',4',5-tetrachlorobiphenyl (tetraCB) (IU-70), 2,2',3',4',5-pentaCB (IU-87), 2,2',4',5,5'-pentaCB (IU-101) and 2,2',3',4',5,5'-hexachlorobiphenyl (hexaCB) (IU-141), and their MeSO2 metabolites were investigated in rats. Administration at various doses (0.2-1.0 mumol/kg) of 3-MeSO2-2,2',4',5,5'-pentaCB produced nearly dose-related increases in the hepatic concentration of this methyl sulphone, in the contents of cytochromes P-450 and b5, and in activities of aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and benzo[a]pyrene hydroxylase of liver microsomes. Major PB-inducible forms, CYP2B1, CYP2B2, CYP3A2 and CYP2C6 were induced with four PCBs (342 mumol/kg) and their 3-MeSO2 metabolites (0.5-10 mumol/kg), indicating that 3-MeSO2 metabolites were strong PB-type inducers of hepatic drug-metabolizing enzymes. 3-MeSO2-2,2',4',5,5'-pentaCB was an especially strong inducer. On the other hand, four PB-inducible forms of cytochrome P-450 were not induced with the 4-MeSO2 isomers. The relation between liver concentrations of the corresponding 3-MeSO2 derivatives and induction of four PB-inducible forms of cytochrome P-450 after administration of four PCBs and their 3-MeSO2 derivatives further confirmed that the 3-MeSO2 metabolites played an important role in the induction which parent PCB congeners caused on the hepatic drug-metabolizing enzyme system.

Animals

Cloning and sequencing of a human endothelin converting enzyme in renal adenocarcinoma (ACHN) cells producing endothelin-2.

Endothelin (ET)-2 is a 21 residue vasoactive peptide which is biosynthesized from big ET-2(1-38) by a specific cleavage at Trp21-Val22 with an ET converting enzyme (ECE). To identify an ECE in ACHN (human renal adenocarcinoma) cells which produce ET-2, we have cloned and sequenced a novel cDNA encoding a human ECE in ACHN (hAECE). It encodes a 770 amino acid protein with a zinc-binding motif and a single membrane spanning region. The sequences of nucleic acids and amino acids from Leu45 to Trp770 of hAECE are identical to those from Leu33 to Trp758 of a human ECE in HUVEC (hHECE). The sequences in the amino-terminal moiety are divergent between hAECE and hHECE. Based on the difference of the amino-terminal amino acid sequences, ECEs reported so far, can be classified into two isoforms. These results strongly suggest that an alternative splicing might occur in the 5'-terminal region of the ECE pre-mRNA.

Adenocarcinoma

AF64A disrupts retrieval processes in long-term memory of mice.

Intracerebroventricular (i.c.v.) injection of ethylcholine mustard aziridinium ion (AF64A) in mice caused a reversible impairment of retrieval processing in long-term memory. After long retention intervals (1-3 weeks), AF64A-treated mice demonstrated marked impairment of behavioural performance previously acquired in a complex multiple maze task. This behavioural deficit was dose dependently ameliorated by the administration of oxotremorine. In parallel with the behavioural deficit, AF64A selectively decreased acetylcholine (ACh) levels in the hippocampus. These findings suggest that the reversible behavioural deficit induced by AF64A should be regarded as a retrieval failure in long-term memory, and not as a retention failure. This failure was largely associated with dysfunction of the cholinergic neuronal system in the hippocampus.

Animals

In vivo evidence for non-universal usage of the codon CUG in Candida maltosa.

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNASerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.

Amino Acid Sequence

Pancreatic enlargement in obstructive jaundice. Effects of biliary stream diversion in humans.

To verify the influence of obstructive jaundice on pancreatic growth, the anteroposterior width of the pancreas was measured by computed tomography in 30 cholangiocarcinoma patients excluded patients with distal bile duct tumor (jaundice group) and 74 control subjects. Follow-up examinations were performed on 12 patients with and without internal biliary drainage to elucidate the temporal relationship between pancreatic enlargement and the diversion of the obstructed biliary stream. Histologic analysis on autopsy samples from 13 control and 10 jaundice cases also was performed. Mean pancreatic head and body widths in the jaundice group were 2.93 +/- 0.3 cm and 2.01 +/- 0.3 cm, respectively. These values were significantly greater than those of the controls (2.13 +/- 0.3 cm and 1.49 +/- 0.3 cm, P < 0.01). The glandular widths returned to their normal sizes following internal biliary drainage. No changes were seen in patients who underwent external drainage alone. Histologic examination revealed that enlargement of the acinar cells or of the islet of Langerhans was often seen in the jaundiced patients. Therefore obstructive jaundice is thought to cause pancreatic growth through a trophic effect by interrupting biliary circulation.

Aged

Abnormalities in elastic fibers and other connective-tissue components of floppy mitral valve.

Histologic, immunohistochemical, and ultrastructural studies were performed on 12 floppy mitral valves, 4 mitral valves showing focal myxomatous changes without prolapse, and 3 normal mitral valves. All floppy mitral valves were thickened by deposits of proteoglycans and also showed diverse structural abnormalities in collagen and elastic fibers. From these observations we conclude that (1) the structure of all major components of connective tissue in floppy mitral valves is abnormal; (2) alterations in collagen and accumulations of proteoglycans are nonspecific changes that may be caused by the abnormal mechanical forces to which floppy mitral valves are subjected because of their excessively large surface area; (3) the presence of excessive amounts of proteoglycans may interfere with the normal assembly of collagen and elastic fibers; (4) abnormalities of elastic fibers resemble those in other conditions characterized by structural dilatation or tissue expansion; and (5) alterations in elastin could result from defective formation, increased degradation, or both.

Actin Cytoskeleton

Basal levels of noradrenaline, dopamine, 5-hydroxytryptamine, and acetylcholine in the submandibular, parotid, and sublingual glands of mice and rats.

The salivary glands of 5-week-old mice and rats were divided into submandibular, parotid, and sublingual and analysed to determine basal levels of the neurotransmitters noradrenaline (NE) and acetylcholine (ACh) and the possible neurotransmitters dopamine (DA) and 5-hydroxytryptamine (5-HT), using a combination of high-performance liquid chromatography coupled with coulometric and amperometric detection and a direct injection technique employing crude homogenate supernatants. In both species, levels of NE were higher in the submandibular than in the other salivary glands, whereas levels of ACh were higher in the mouse submandibular and the rat sublingual glands than in other glands. In all the salivary glands, levels of DA were markedly lower than those of other target substances. Levels of 5-HT were similar in all salivary glands. These results show that in mouse and rat salivary glands, species differences in neurotransmitter distribution are relatively small, whereas there are considerable differences in distribution between the salivary glands.

Acetylcholine

The dopaminergic system modulates the endogenous opioid system in guinea-pig isolated ileal longitudinal muscle.

The effects of the dopamine antagonists haloperidol and sultopride were investigated on the twitch response, evoked by 0.1 Hz stimulation of guinea-pig isolated ileal longitudinal muscle, and on the inhibition of the twitch response induced by 10 Hz stimulation (post-tetanic twitch inhibition) and by application of opioids. Both haloperidol and sultopride concentration-dependently inhibited the twitch response, with threshold concentrations of 2 and 50 microM, respectively, and could also shift the concentration-response curve for ACh-contraction to the right in a non-competitive manner. Haloperidol (1 microM) and sultopride (20 microM) increased post-tetanic twitch inhibition and this could be prevented by naloxone (100 nM). Twitch inhibition induced by morphine and dynorphin 1-13 was not affected by haloperidol (1 microM) or sultopride (20 microM). Prazosin (1 microM) and yohimbine (2 microM) did not affect either the twitch response or the post-tetanic twitch inhibition. These results suggest that dopamine receptors are involved in the modulation of the ileal opioid system, in such a manner as to diminish the release of endogenous opioids by tetanic stimulation.

Amisulpride

Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells.

A low concentration of bufalin, a component of bufadienoides in the traditional Chinese medicine chan'su, was shown previously to induce differentiation of a broad range of human leukemia cell lines. In the present study, we found that bufalin at concentrations of 10(-7) M and higher induced apoptosis in human leukemia cells, such as HL60, ML1, but not in mouse leukemia M1 cells. A mere 15 min pretreatment of HL60 cells with 10(-6) M bufalin, followed by incubation for 15 h without bufalin, caused fragmentation of DNA and a decrease in cell viability, indicating that the signal for induction of apoptosis is triggered rapidly upon treatment with bufalin. Bufalin-induced apoptosis in HL60 cells was inhibited by ZnCl2, an inhibitor of endonuclease, but not by cycloheximide, an inhibitor of protein synthesis. Northern blot analysis revealed that the levels of expression of the c-myc and bcl-2 genes in HL60 cells decreased with time after treatment with bufalin. These results suggest that bufalin induces apoptosis specifically in human leukemia cells by altering the expression of these genes involved in apoptosis.

Animals

Cardiovascular selectivity of 1,4-dihydropyridine derivatives, efonidipine (NZ-105), nicardipine and structure related compounds in isolated guinea-pig tissues.

1. The cardiovascular selectivities of 1,4-dihydropyridine derivatives, efonidipine (NZ-105), nicardipine, 3NZ5NIC (the drug with NZ-105-type side-chain at C3 position and nicardipine-type at C5) and 3NIC5NZ (the drug with nicardipine-type side chain at C3 and NZ-105-type at C5) were studied in vitro. 2. All four compounds caused relaxation of guinea-pig aortae precontracted with a high K+. The pEC50 values were 7.5, 8.3, 8.1 and 5.6, for NZ-105, nicardipine, 3NIC5NZ and 3NZ5NIC, respectively. The relaxation produced by NZ-105 was slower in onset than those produced by the other compounds. The rate constant K(hr-1) of the relaxations were 0.59, 1.31, 1.02 and 1.24, for NZ-105, nicardipine, 3NIC5NZ and 3NZ5NIC, respectively. 3. In the electrically paced guinea-pig papillary muscles, NZ-105, 3NIC5NZ and 3NZ5NIC, even at concentrations as high as 10(-6) M, slightly decreased the contractile force (by 44.9 +/- 7.1%, 58.6 +/- 5.4% and 52.2 +/- 3.9%, respectively), whereas 10(-6) M nicardipine decreased the force by 84.9 +/- 3.3%. The negative inotropic effect of NZ-105 and 3NIC5NZ, but not that of 3NZ5NIC or nicardipine, was over 10 times weaker than their vasorelaxant effect. 4. In the guinea-pig right atria, NZ-105 and nicardipine at 10(-8) M decreased the spontaneous contraction rate by 67.9 +/- 15.0% and 39.7 +/- 15.4%, respectively. 3NIC5NZ at 3 x 10(-9) M and 3NZ5NIC at 3 x 10(-8) M had little effect on the rate, whereas 10(-8) M 3NIC5NZ and 10(-7) M 3NZ5NIC arrested the beating within 3 hr after administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Effects of efonidipine hydrochloride (NZ-105), a calcium antagonist, on renal function in conscious spontaneously hypertensive rats.

1. We investigated the effects of short- and long-term administration of efonidipine hydrochloride (NZ-105), 1,4-dihydropyridine derivative, in conscious spontaneously hypertensive rats (SHR). 2. Oral administration of NZ-105 for 12 weeks caused diuretic and natriuretic effects, which were not attenuated during the experimental period. 3. In the short-term experiment for investigating the mechanism of the diuretic effect, intravenous injection of NZ-105 (0.03 mg/kg of body weight) significantly increased the urine volume (UV), renal plasma flow (RPF) and glomerular filtration rate (GFR). The increment rate of UV and RPF was 105.4 +/- 17.8% and 111.7 +/- 72.8%, respectively, which were larger than the increment rate of GFR (38.5 +/- 14.0%). 4. The diuretic or natriuretic effect of NZ-105 was suggested to be due to both the inhibition of sodium reabsorption and, at least in part, the increase of GFR.

Animals

Direct evidence for the occurrence of superoxide radicals in the small intestine of the burned rat.

To determine if superoxide radicals (O2-) and related metabolites are generated in extradermal tissues of burned animals, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazol [1,2-å]pyrazin-3-one (MCLA) was infused intravenously into rats, and change in the chemiluminescence (CL) intensity of the small intestine was determined by using a sensitive photodetector. When animals were challenged with burn stress of 40% total body surface area (TBSA), the CL intensity of the intestine gradually increased, reaching a maximum within 1 hour and remaining elevated for up to 3 hours. Pretreatment of animals with a long-acting superoxide dismutase (SOD) derivative (SM-SOD) significantly inhibited the increase in CL intensity. Administration of SM-SOD immediately after inducing burn injury also significantly inhibited the increase in CL. These results suggest that superoxide radicals are generated in extradermal tissues, such as the small intestine, in the early stage after burn injury.

Animals

Demonstration of Epstein-Barr virus genomes, using polymerase chain reaction in situ hybridization in paraffin-embedded lymphoid tissues.

We used the polymerase chain reaction (PCR) in situ hybridization (ISH) (PCR-ISH) on sections of malignant lymphoma and nonspecific lymphadenitis to detect small amounts of Epstein-Barr virus (EBV), a DNA virus of the herpes virus family. We first surveyed the EBV DNA by Southern blot analysis and PCR, and then compared results of the two PCR/ISH methodologies with the results of simplified/sensitive ISH for the positive cases. The target of the simplified in situ (DNA-ISH) was a few copies of EBV DNA per cell, and the target of the sensitive in situ (RNA-ISH) was as many as 10(7) copies of EBV RNA per cell. When EBV DNA was detected by Southern blot, DNA-ISH, RNA-ISH and PCR-ISH all revealed EBV genomes. When PCR revealed only amplified EBV DNA, DNA-ISH showed no EBV genomes, but PCR-ISH and RNA-ISH showed EBV genomes in a few cells. When PCR showed no detectable amplified EBV DNA, all of DNA-ISH, RNA-ISH and PCR-ISH showed no genomes. These findings indicate that PCR-ISH consistently detected a few copies of the EBV virions. The PCR-ISH was as sensitive as RNA-ISH. The RNA-ISH could not detect virus if RNA was not expressed, but the PCR-ISH could detect virus without such expression. The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology, and cell biology.

Base Sequence