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Biomedical subjects

K Haga

Publications and source records attributed to K Haga.

At least 55 records · Page 3Linked to original sources
PubMed1995

Surface-Sensitive XAFS in the Hard X-ray Region with Sub-Monolayer Sensitivity.

Surface-sensitive X-ray absorption fine structure (XAFS) with sub-monolayer sensitivity based on grazing-incidence fluorescence detection is reported. The efficiency of fluorescence detection increased by more than two orders of magnitude by combining a multipole wiggler with a multi-element Si(Li) solid-state detector. The capability of the present technique for structural studies of surfaces and buried interfaces in the hard X-ray region was demonstrated by As K-edge XAFS studies of the InP(001) surface exposed to AsH(3) flow. The results indicated that ~0.1 monolayer As atoms are incorporated into the surface replacing the P atoms.

Journal Article↗
PubMed1995

The function of 5-HT3 receptors on colonic transit in rats.

The function of serotonin (5-HT)3 receptors on colonic transit was investigated in unanesthetized rats. The colonic transit was accelerated by 5-HT (10 mg/kg, s.c.), 2-methyl-5-HT (30 mg/kg, s.c.), neostigmine (0.03-0.1 mg/kg, s.c.), corticotropin releasing factor (CRF; 1 microgram intracerebroventricular administration) and restraint stress (for 45 minutes). A potent and selective 5-HT3 receptor antagonist, azasetron (+/-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro- 4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide monohydrochloride ; 0.01-10 mg/kg, p.o. inhibited the 5-HT-, CRF- and stress-accelerated colonic transit in a dose-dependent manner. Ondansetron (10 mg/kg, p.o.) and granisetron (1 mg/kg, p.o) also inhibited the stress-accelerated colonic transit, but azasetron was more effective than these two drugs. Atropine methylbromide (0.1 mg/kg, s.c.) and tetrodotoxin (0.01 mg/kg, s.c.) inhibited the accelerated colonic transit under stress conditions, but methysergide (10 mg/kg, s.c.), SDZ205-557 (10 mg/kg, s.c.), domperidone (30 mg/kg, p.o.), trimebutine (300 mg/kg, p.o.), did not. Azasetron (10 micrograms) administered intracerebroventricularly did not inhibit the stress-induced acceleration. These results suggest that endogenous 5-HT which is released through stress accelerates the colonic transit via the 5-HT3 receptors and finally a cholinergic mechanism. It is considered that azasetron inhibits colonic transit particularly under stress conditions through the blockade of the peripheral 5-HT3 receptors. Azasetron may improve bowel function in stress-related colonic dysfunction like irritable bowel syndrome.

Animals↗
PubMed1995

Relationship between the phasic period of interdigestive migrating contraction and the systemic bioavailability of acetaminophen in dogs.

The relationships of the phasic period of interdigestive migrating contraction to gastrointestinal (GI) transit of drugs and their oral absorption were investigated in mongrel dogs by simultaneous oral dosing of acetaminophen (AAP) and salicylazosulfapyridine (SASP) at the starting points of the phase I and phase III periods of gastric contractions. Strain-gauge force transducers were surgically sutured onto the serosa of the GI tracts in the dogs to measure the interdigestive migrating contractions. The mean absorption time of AAP and the time for the first appearance of sulfapyridine (a bacterial metabolite of SASP in the colon) in plasma were used as the indices of gastric emptying time (GET) and small intestinal transit time (SITT), respectively. In individual dogs, the GET and the SITT at phase I showed a clear delay in comparison with those at phase III. For AAP used as a marker compound here, the systemic bioavailability after oral dosing to intact beagle dogs at doses of 3, 10, and 20 mg/kg was about 55, 63, and 79%, suggesting that AAP undergoes a non-linear hepatic clearance. At a dose of AAP 20 mg/kg, the systemic bioavailability of AAP was 100% in the case of dosing at phase III, but was reduced by half when dosing at phase I. These results indicate that, in oral dosing, the transit of drugs through the GI tract was clearly affected by the phases of gastric contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗
PubMed1995

[Cisapride, a gastroprokinetic agent, binds to 5-HT4 receptors].

The affinity of cisapride for 5-HT4 receptors was investigated in comparison with those of other 5-HT4 receptor agonists and antagonists, such as 5-HT, 5-MeOT, mosapride, zacopride, metoclopramide, BIMU8, GR113808, SDZ 205-557 and ICS 205-930. Cisapride and the other compounds dose-dependently inhibited specific 3H-GR113808 binding to 5-HT4 receptors in guinea pig striatal membranes, and complete inhibition of specific 3H-GR113808 binding was achieved at the high concentrations of these compounds. Cisapride was 1.9-, 7.3-, 4.3-, 11- and 26-fold more potent than 5-HT, 5-MeOT, mosapride, zacopride and metoclopramide, respectively, in competing for 5-HT4 receptors. To determine the manner of interaction between cisapride and 5-HT4 receptors, Scatchard analysis of 3H-GR113808 specific binding to striatal membranes was performed. Cisapride increased the Kd value of 3H-GR113808 in striatal membranes in a dose-dependent manner without any influence on the binding density (Bmax) of 3H-GR113808. These findings indicate that cisapride binds to 5-HT4 receptors competing with 3H-GR113808 in guinea pig striatal membranes.

Animals↗
PubMed1995

[Quantitative autoradiographic analysis of the binding of mosapramine to dopamine D3-receptors].

The affinity of mosapramine, an iminodibenzyl antipsychotic, to dopamine D3-receptors in rat brain was investigated by quantitative autoradiography of [3H]-7-OH-DPAT, a selective D3-ligand. Autoradiograms showed restricted distribution of [3H]7-OH-DPAT binding sites, with very high levels on the islands of Calleja (ICj), olfactory tubercle (Tu) and nucleus accumbens, while low but distinct labeling was observed in the molecular layer of lobule 10 of the cerebellum and caudate putamen (CPu). Binding of [3H]7-OH-DPAT completely disappeared when 1 microM dopamine was added, and it was reduced by the addition of mosapramine in a concentration-dependent manner. The displacing effect of mosapramine was more potent than that of haloperidol or clozapine in the brain regions examined. Mosapramine showed more potent affinity to receptors in Tu and ICj than those in CPu. On the other hand, haloperidol and clozapine did not show such regional differences. These results suggest that the high affinity of mosapramine to D3-receptors participates in, at least in part, the development of the clinical effects of mosapramine.

Animals↗
PubMed1995

[Age estimation by appearance of gray hair in pubic hair].

The pubic hair of Japanese males aged 12-90 years old (n = 123) and females 14-85 ys. (n = 80) autopsied during 1984-1993 was investigated to determine its appearance age and possible appearance age of gray hair. As the appearance degree of gray hair in pubic hair 5 stages were applied to samples to establish a criterion for age estimation. Stage 0; no gray hair, 1; 1-3 gray hairs, 2; a few-10, 3; 10 gray hairs-2/3 of the entire pubic hair, 4; more than 3/4. The appearance age of gray hair with appearance rate 100% (75%) was more than 65 (55) ys. in both sex. The possible appearance age was more than 30 ys. in males and 36 ys. in females, practically younger than "45 years old" which had been described in texts. The age range with a hit rate 100% is at stage 0; less than 62 ys. in males, less than 59 ys. in females, at stage 1; 30-64 ys. in males, 36-60 ys. in females, at stage 2; 43-72 ys., 39-85 ys. and at stage 3; 49-77 ys,--.

Adolescent↗
PubMed1995

[An autopsy case of a pacemaker implanted patient].

A pacemaker implanted patient (female, 78 years old) was found dead in her bed and judicially autopsied. Before postmortem inspection the pacemaker was removed by a doctor. We report the problems in such as the doctor's treatment, additionally accommodation of medical examiners and police, and determination of a cause of death in autopsy.

Aged↗
PubMed1995

[Relationship among "kenshi" (inquest), "kenshi" (postmortem examination) and "ken-an" (postmortem inspection) in Japan].

Inquest ([symbol: see text]) and postmortem examination ([symbol: see text]) have the same pronunciation of "kenshi" in Japan. So we are inclined to use these terms with confusion or substitution. We discussed the differences among three "kenshis" adding to postmortem inspection ([symbol: see text]; "ken-an") with investigating references. Many forensic scientists described that inquests were done by public prosecutors and inspections by medical doctors. But there were various interpretations of postmortem examination, e.g. "postmortem examination is done by public prosecutors or police", "it is the same as inspection by medical doctor", "it is one of the aids of inquest", "it is also including forensic autopsy". Although being no differences between the two kenshis written by two kinds of different Chinese characters ([symbol: see text]). This is caused an only problem in writing Japanese languages, it happens especially when we have to use Chinese characters for everyday use. In order to define this postmortem examination in Japan, we paid attention to corpse procedures. We made a figure to include each interpretation and to understand that postmortem examination had several meanings from a narrow sense to a wide sense. We suggested that the most confident definition was of the widest sense which meant the inspection and autopsy by doctors and the inspection by public prosecutors or police.

Forensic Medicine↗
PubMed1994

Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

Animals↗
PubMed1994

Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization.

The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.

Amino Acid Sequence↗
PubMed1994

Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics.

Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed. Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate. Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.

Acarbose↗
PubMed1994

Synergistic activation of a G protein-coupled receptor kinase by G protein beta gamma subunits and mastoparan or related peptides.

Muscarinic acetylcholine receptors (mAChR, m2 subtype) are phosphorylated in an agonist-dependent manner by a G protein-coupled receptor kinase (GR kinase) at sites located in the central part of mAChR. The GR kinase phosphorylated a glutathione S-transferase fusion protein containing the m2 phosphorylation sites (13-GST). Phosphorylation of 13-GST was markedly stimulated by mastoparan: the Km for 13-GST decreased from 7.3 to 0.17 microM in the presence of 100 microM mastroparan and the Vmax increased 23-fold. beta gamma Subunits of G proteins synergistically stimulated the phosphorylation of 13-GST in the presence of 1-10 microM mastoparan by increasing the affinity of mastoparan for the GR kinase. The phosphorylation of mAChR by the GR kinase was also synergistically stimulated by 1-10 microM mastoparan in the presence of beta gamma subunits, but was inhibited by 100 microM mastoparan. Similar stimulatory and inhibitory effects on the phosphorylation of 13-GST and mAChR were observed using synthetic peptides of 15-20 amino acid residues corresponding to sequences of intracellular segments adjacent to the transmembrane segments of mAChR. These results are consistent with and support the idea that the GR kinase is synergistically activated by both G protein beta gamma subunits and agonist-bound receptors.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray studies of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011.

Large crystals of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. 1011, a typical alkalophilic enzyme, have been obtained at room temperature using polyethylene glycol 3000 and 2-propanol as precipitant. They belong to the triclinic space group P1 with the following unit cell constants: a = 64.93 A, b = 74.45 A, c = 79.12 A, alpha = 85.2 degrees, beta = 105.0 degrees and gamma = 101.0 degrees. The crystallographic asymmetric unit seems to contain two molecules of CGTase, with crystal volume per protein mass (Vm) of 2.41 A3/Da and solvent content of 49% by volume. The crystals diffract to at least 2.0 A resolution and they are suitable for X-ray analysis.

Bacillus↗
PubMed1994

Location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype).

Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist-dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled bands decreased following treatment with N-glycanase. The 59-kDa and 39-kDa bands were converted to 52-kDa and 32-kDa bands, respectively, indicating that both the 59-kDa and 39-kDa bands contain the amino-terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59-kDa and 39-kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino-terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10-11/receptor, with 7-8 serine and 3-4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30-kDa amino-terminal segment. These results indicate that the phosphorylation sites are localized in the range 30-39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S-transferase linked to a peptide corresponding to residues 227-324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin-sensitive manner.

Amino Acid Sequence↗
PubMed1994

The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping-Pong mechanism.

A new photometric assay of the disproportionation activity of cyclodextrin glucanotransferase (CGTase) using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl-maltopentaoside as the donor, proved that the transglycosylation reaction of CGTase was operated by a Ping-Pong Bi Bi mechanism. The values of the kcat/Km(acceptor) proved that the same configurations of free hydroxyl groups with those of D-glucopyranose at C2, C3 and C4 positions were required for the acceptors used by CGTase. The structure around C6 on acceptors was not essential for acceptor function, but it was recognized by CGTase, since the values of kcat/Km for D-xylose were smaller than that for D-glucose. The value of kcat/Km for maltose was about 20-times larger than that for D-glucose, indicating that at least two glucopyranosyl rings are recognized by the acceptor binding sites.

Bacillus↗
PubMed1994

Estimation of time of death by quantification of melatonin in corpses.

A method for the estimation of time of death (TOD), was evaluated by measuring the melatonin (MT) content of pineal bodies (PBs), sera and urine samples from 85 cadavers. A total of 44 cadavers were investigated in Sapporo (geographical coordinates N 43 degrees 4', E 141 degrees 21') and 41 in Tokyo (N 35 degrees 39', E 139 degrees 44'). MT contents were measured by radioimmunoassay (RIA) in 75 PBs, 27 sera and 14 urine samples. Exponential differences of pineal MT content were recognized between peaks in nighttime and nadirs in daytime, ranging from 0.099 to 63.2 ng/PB. Circadian rhythms were also observed for the concentrations of MT in serum (11-205 pg/ml), and in urine (7.5-137.5 pg/ml). Consequently, criteria for the TOD estimation are proposed as follows. 1) Pineal MT contents--(1) 0-0.2 ng/PB: TOD 1100-1700 hours, (2) 0.2-0.3 ng/PB: TOD 0700-2000 hours, (3) 0.3-1 ng/PB: inconclusive, (4) 1-4 ng/PB: TOD 1600-1000 hours, (5) 4-8 ng/PB: TOD 2000-0800 hours, (6) over 8 ng/PB: TOD 2000-0500 hours, 2) Serum MT concentration--(1) 0-100 pg/ml: inconclusive, (2) over 100 pg/ml: TOD 2200-0100 hours, and 3) Urinary MT concentration--(1) 0-35 pg/ml: inconclusive, (2) over 35 pg/ml: TOD 1800-0600 hours. The range of the estimation can be limited by a combination of these 3 criteria. The present method can be combined with other methods for estimating the TOD to decrease the range.

Adolescent↗
PubMed1994

A role of iron in lambda DNA strand breaks in the reaction system of alloxan with reduced glutathione: iron(III) binding to the DNA.

lambda DNA strand breaks were easily induced in a reaction system involving alloxan with reduced glutathione (GSH) in the presence of FeCl3 in a HEPES-NaOH buffer, pH 7.4. Increasing concentrations of FeCl3 in the reaction system caused DNA strand breaks in a concentration-dependent fashion, suggesting that iron is required to induce the DNA strand breaks. Catalase, scavengers of hydroxyl radicals (HO.) and iron-chelators almost completely inhibited the DNA strand breaks, but superoxide dismutase (SOD) did not do so, suggesting that the HO., formed by a Fenton-type reaction, was the species responsible for the DNA strand breaks. The addition of FeCl3 to the solution containing DNA caused the formation of a DNA-Fe(III) complex, in which Fe(III) was reduced by an alloxan radical (HA.) but not by a superoxide radical. Only when apotransferrin was added to the reaction mixtures before the addition of FeCl3, were both the DNA strand breaks and the reduction of Fe(III) strongly inhibited. These results suggest that the Fe(III) bound to DNA catalyzes the DNA strand breaks which may be caused by the generation of site-specific HO. via an HA.-dependent Fenton-type reaction.

Alloxan↗