Biomedical subjects

Yukihiro Shoyama

Publications and source records attributed to Yukihiro Shoyama.

At least 19 recordsLinked to original sources

PubMed2007

Kamikihi-to, a Kampo medicine, ameliorates impairment of spatial memory in rats.

The present study investigated the effects of Kamikihi-to (KKT), a Kampo medicine, on impairment of spatial memory in rats using an eight-arm radial maze task. Scopolamine (0.5 mg/kg, i.p.), a non-selective muscarinic receptor antagonist, and Delta(9)-tetrahydrocannabinol (THC; 6 mg/kg, i.p.), a principal psychoactive component of marihuana, each markedly impaired the spatial memory. KKT (1 and 3 mg/kg, p.o.) significantly improved the scopolamine-induced impairment of spatial memory. KKT (30 mg/kg, p.o.) also improved significantly the THC-induced impairment of spatial memory. Moreover, KKT (3 and 30 mg/kg, p.o.) enhanced tremors induced by oxotremorine, a muscarinic M(1) receptor agonist. Taken together these findings suggest that KKT is a useful drug for treating memory deficits.

Animals↗

PubMed2006

Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo.

Crocus sativus L. (saffron) has been used as a spice for flavoring and coloring food preparations, and in Chinese traditional medicine as an anodyne or tranquilizer. Our previous study demonstrated that crocin, a carotenoid pigment of saffron, can suppress the serum deprivation-induced death of PC12 cells by increasing glutathione (GSH) synthesis and thus inhibiting neutral sphingomyelinase (nSMase) activity and ceramide formation. The carotenoid pigments of saffron consist of crocetin di-(beta-d-glucosyl)-ester [dicrocin], crocetin-(beta-d-gentiobiosyl)-(beta-d-glucosyl)-ester [tricrocin] and crocetin-di-(beta-d-gentiobiosyl)-ester [crocin]. Saffron also contains picrocrocin, the substance causing saffron's bitter taste. In this study, to confirm whether neuroprotective effects of saffron are caused solely by crocin, we examined the antioxidant and GSH-synthetic activities of these crocins in PC12 cells under serum-free and hypoxic conditions. Measurements of cell viability, peroxidized membrane lipids and caspase-3 activity showed that the rank order of the neuroprotective potency at a concentration of 10 muM was crocin>tricrocin>dicrocin and picrocrocin (the latter two crocins had a little or no potency). In addition, we show that among these saffron's constituents, crocin most effectively promotes mRNA expression of gamma-glutamylcysteinyl synthase (gamma-GCS), which contributes to GSH synthesis as the rate-limiting enzyme, and that the carotenoid can significantly reduce infarcted areas caused by occlusion of the middle cerebral artery (MCA) in mice.

Animals↗

PubMed2006

One-step immunochromatographic separation and ELISA quantification of glycyrrhizin from traditional Chinese medicines.

The bioactive constituent, glycyrrhizin or glycyrrhizic acid (GA), was purified from two traditional Chinese medicines (TCM), Shaoyao gancao tang and Dahuang gancao tang, and from crude extracts from licorice roots by means of immunoaffinity chromatography using anti-GA monoclonal antibody (MAb) and was quantified with an enzyme-linked immunosorbent assay (ELISA). Laboratory preparations included the synthesis of conjugate GA-human serum albumin (GA-HSA), the production of anti-GA-MAb, the optimization of the immunoaffinity column packed with the anti-GA-MAb coupled to hydrazide gel and the determination of the GA content in TCM and crude drugs from five different sources by ELISA and high performance liquid chromatography (HPLC). The experimental results reveal that the anti-GA-MAb coupled to Affi-Gel Hz gel results in a coupling efficiency of 95.2%, and the immunoaffinity chromatography gives a mean recovery of 97.6% of GA with a capacity of 33.5+/-2.40 microg/mL of immunoaffinity gel under the given conditions. The GA content of the crude extracts (ranging 74.8-114.6 microg/mg) from different sources by the ELISA method is much greater than that of the TCM (16.4-25.1 microg/mg) which is, in good agreement with the results of the HPLC method. Our report provides a rapid, reliable and sensitive approach for one-step separation and quantification of GA.

Chromatography, Affinity↗

PubMed2006

Involvement of 5-hydroxytryptamine1A receptors in Delta9-tetrahydrocannabinol-induced catalepsy-like immobilization in mice.

The present study investigated the involvement of 5-hydroxytryptamine(1A) (5-HT(1A)) receptors in Delta(9)-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization in mice. THC (10 mg/kg, i.p.) induced catalepsy-like immobilization but had no effect on motor coordination in the rota-rod test. The selective cannabinoid CB(1) receptor antagonist rimonabant (3 mg/kg, i.p.) completely antagonized THC-induced catalepsy-like immobilization. The 5-HT(1A)/5-HT(7) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 0.3 and 1 mg/kg, i.p.) and 5-HT(1A) receptor partial agonist buspirone (0.06 and 0.1 mg/kg, i.p.) inhibited this THC-induced catalepsy-like immobilization. Moreover, the selective 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohezane carboxamide dihydrochloride (WAY100635; 0.3 or 1 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization by 8-OH-DPAT (1 mg/kg) or buspirone (0.06 mg/kg). In contrast, the selective 5-HT(7) receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl]phenol hydrochloride (SB269970) had no effect on this inhibitory effect of 8-OH-DPAT. On the other hand, WAY100635 (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). These findings suggest that the 5-HT(1A) receptors are involved in THC-induced catalepsy-like immobilization.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2006

High-throughput determination of free D-aspartic acid in mammals by enzyme immunoassay using specific monoclonal antibody.

A method for rapid determination of free D-aspartic acid (D-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against D-Asp for the first time. An anti-D-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated D-Asp to BALB/c mice. The obtained antibody has a high specificity toward D-Asp but shows a slight cross-reactivity to all other D- and L- amino acids including L-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 micromol/mL D-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of D-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of D-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of D-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this D-amino acid.

Amino Acid Sequence↗

PubMed2006

Antipsychotics improve Delta9-tetrahydrocannabinol-induced impairment of the prepulse inhibition of the startle reflex in mice.

Recently, cannabinoid receptor agonists have been reported to impair prepulse inhibition (PPI) of the startle reflex. In the current study, we examined the effect of Delta9-tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, on the PPI, and found that THC (10 mg/kg, i.p.) impaired the PPI concomitant with a decrease in the startle response. Antipsychotics such as haloperidol (0.3 mg/kg, i.p.) and risperidone (0.1 mg/kg, i.p.), which are potent dopamine D2 receptor antagonists, and SR141716 (10 mg/kg, i.p.), a CB1 cannabinoid receptor antagonist, reversed these THC-induced PPI deficits. Moreover, THC (10 mg/kg) increased dopamine (DA) release in the nucleus accumbens but not medial prefrontal cortex over a 50-100-min period (time of PPI test) after treatment, and SR141716 (10 mg/kg) reversed this increase in DA release induced by THC. These results suggest that dopaminergic hyperfunction in the nucleus accumbens may be involved in THC-induced PPI deficits.

Acoustic Stimulation↗

PubMed2006

Activation of a refolded, berberine-specific, single-chain Fv fragment by addition of free berberine.

A single-chain variable fragment (scFv) specific for berberine was produced in Escherichia coli. The anti-berberine scFv gene was cloned from hybridoma 1D5-3B-7 producing the monoclonal antibody. The variable regions of the heavy (V(H)) and light chain (V(L)) genes were connected with a flexible linker using an assembly PCR. The V(H)-linker-V(L) gene was inserted into a plasmid, pET28a (+), then overexpressed in E. coli BL21 (DE3). The active of the scFv by refolding based on stepwise dialysis methods and an artificial chaperone was determined by direct and competitive enzyme-linked immunosorbent assay (ELISA). The results of direct ELISA showed that the anti-berberine scFv retained specific binding activity to berberine. In competitive ELISA, however, activity was increased depending on the concentration of berberine.

Animals↗

PubMed2006

Visual detection of saikosaponins by on-membrane immunoassay and estimation of traditional Chinese medicines containing Bupleuri radix.

The purpose of this study was to describe the simple, rapid, and environmental-cost effective determination method for saikosaponins in complicated samples like Bupleuri radix and traditional Chinese medicines (TCM). Saikosaponin standards, extracts of Bupleuri radix and TCM, were applied to a polyethersulphone (PES) membrane and developed by acetonitrile-water (1:4, by volume). Saikosaponin a (SSa), SSc, and SSd were visually detected by an immunostaining method (called Eastern blotting technique) using a monoclonal antibody (MAb) against SSa. At least 62.5 ng of SSa, SSc, and SSd were clearly detectable individually. These coloring spot areas of saikosaponins on PES membrane were calculated by using the NIH Imaging software and three saikosaponins can be analyzed quantitatively between 62.5 ng and 1.0 microg. Saikosaponins in Bupleuri radix and TCM were determined and these results of SSa and total saikosaponin concentrations were in good agreement with those from the ELISA analysis.

Bupleurum↗

PubMed2006

Low dose citalopram reverses memory impairment and electroconvulsive shock-induced immobilization.

Citalopram, a selective serotonin reuptake inhibitor (SSRI), is one of the most widely used antidepressants. Recently, citalopram has been reported to improve working memory in patients with depression, and psychotic symptoms and behavioral disturbances in patients with dementia. However, the possibility of using citalopram in the treatment of cognitive disorders has not received much attention. The present study investigated the effects of citalopram on scopolamine- and Delta9-tetrahydrocannabinol (THC)-induced impairment of spatial memory using an eight-arm radial maze and electroconvulsive shock (ECS)-induced immobilization (a behavioral model for the disturbance of consciousness). Low dose citalopram reversed both scopolamine- and THC-induced impairment of spatial memory, suppressed ECS-induced immobilization reversed the THC-induced decrease of acetylcholine (ACh) release in the dorsal hippocampus in vivo microdialysis, and enhanced tremors induced by oxotremorine, a muscarinic M1 receptor agonist. Taken together these findings suggest that low dose citalopram is useful for the treatment of memory deficits and consciousness disturbance.

Animals↗

PubMed2006

Antiviral flavonoid-type C-glycosides from the flowers of Trollius chinensis.

Two new flavonoid-type C-glycosides, trollisin I (= (1S)-1,5-anhydro-1-[2-(3,4-dihydroxyphenyl)-5-hydroxy-7-methoxy-4-oxo-4H-[1]benzopyran-8-yl]-2-O-(2-methylbutanoyl)-D-glucitol; 1) and its 2-O-benzoyl congener trollisin II (2), were isolated from Trollius chinensis Bunge, together with the two known compounds 2''-O-(2'''-methylbutanoyl)isoswertisin (3) and vitexin galactoside (4). All compounds were identified by HR-ESI-MS and in-depth NMR-spectroscopic analyses. In antiviral assays, compound 3 was found to be moderately active towards influenza virus A.

Animals↗

PubMed2006

Construction and expression of a single chain Fv fragment against pharmacologically active paeoniflorin in Escherichia coli, and its potential use in an enzyme-linked immunosorbent assay.

A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (V (H)) and light (V (L)) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly (4)Ser) (3) linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 microg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.

Antibodies, Monoclonal↗

PubMed2006

Pharmacokinetic study of ginsenosides Rb1 and Rg1 in rat by ELISA using anti-ginsenosides Rb1 and Rg1 monoclonal antibodies.

Enzyme-linked immunosorbent assay (ELISA) systems using anti-ginsenoside Rb1 (G-Rb1) and Rg1 (G-Rg1) monoclonal antibodies (MAbs) were established for pharmacokinetic investigations of G-Rb1 and G-Rg1 in rat serum. The systems not only allowed sensitive detection of G-Rb1 at the level as low as 20 ng/ml and of G-Rg1 at 300 ng/ml, but showed strong capacity for detecting the two agents in a broad concentration range (20 to 400 ng/ml for G-Rb1 and 0.3 to 10 microg/ml for G-Rg1, respectively). In this respect, these assay systems are superior to other methods using thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). In addition, another advantage of these immunoassays is the comparably low quantities of specimen required; as little as 5 microl of serum suffices the need for determination of ginsenosides. We report in this article the application of this immunoassay in pharmacokinetic study of G-Rb1.

Animals↗

PubMed2005

Decrease in prostaglandin level is a prerequisite for the expression of cannabinoid withdrawal: a quasi abstinence approach.

Cannabinoid withdrawal has been indicated in both human and animal subjects. One of pathways proposed to facilitate cannabinoid action is the arachidonic acid cascade. Previously, we have shown that prostaglandin attenuated the expression of withdrawal signs in tetrahydrocannabinol-dependent mice. It follows that the cascade might participate in the expression of cannabinoid withdrawal. We utilized a quasi abstinence approach (the induction of a state of cannabinoid withdrawal without giving any cannabinoid substances in a naïve animal) to describe the relationship between the change in prostaglandin level, an end product of the arachidonic acid cascade, and the expression of cannabinoid withdrawal. Administration of 10 mg/kg diclofenac, a prostaglandin synthesis inhibitor, i.p. 30 min before SR 141716A induced cannabinoid withdrawal signs in naïve mice, which were comparable to the true abstinence in cannabinoid-tolerant mice. In turn, 10 mg/kg Delta(8)-THC i.p., given 15 min prior to SR 141716A, blocked the expression of these signs. These results suggested that the decrease in prostaglandin level is a prerequisite for the expression of cannabinoid withdrawal.

Animals↗

PubMed2005

Detection and quantification of ginsenoside Re in ginseng samples by a chromatographic immunostaining method using monoclonal antibody against ginsenoside Re.

A chromatographic immunostaining method has been developed for the determination of ginsenoside Re (G-Re) in ginseng samples on a polyethersulphone (PES) membrane. G-Re standard and the extracts of ginseng roots were applied to a PES membrane and developed by methanol-water-acetic acid (45:55:1, by volume). G-Re was clearly detected by an immunostaining method using a monoclonal antibody against G-Re. The coloring spots of G-Re were analyzed quantitatively using NIH Image software indicating at least 0.125 microg of G-Re was detectable. G-Re can be analyzed quantitatively between 0.25 and 4.0 microg.

Animals↗

PubMed2005

An on-membrane quantitative analysis system for glycyrrhizin in licorice roots and traditional Chinese medicines.

An on-membrane quantitative analysis system has been developed for determining glycyrrhizin (GC) in licorice roots and traditional Chinese medicines. A GC standard and the extracts of licorice roots and traditional Chinese medicines were applied to a polyethersulfone (PES) membrane and were developed by acetonitrile/water/formic acid (45:55:2, by volume), then treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a GC-BSA conjugate on a PES membrane. Anti-GC monoclonal antibody was bound and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted with the enzyme and gave staining. The stained membrane was scanned and coloring spots were analyzed quantitatively using graphic analysis by NIH Image software, indicating at least 0.5 microg of GC was clearly detectable. GC can be analyzed quantitatively between 1.0 and 8.0 microg.

Chromatography, High Pressure Liquid↗

PubMed2005

Crystallization of Delta1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa.

Delta1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure-function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A. The calculated Matthews coefficient was approximately 4.1 or 2.0 A3 Da(-1) assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

Cannabis↗

PubMed2005

Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes.

Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.

Base Sequence↗

PubMed2005

Chromatographic resolution of glucosidic compounds, ginsenosides on polyethersulphone membrane, and its application to the quantitative immunoassay for ginseng saponins.

A method has been devised for the chromatographic resolution of glucosidic compounds, ginseng saponins, on polyethersulphone (PES) membrane. The method results in good resolution and quantitative immunoassay for ginsenoside Rb1 (G-Rb1), G-Rc, and G-Rd in crude extracts of various ginsengs. The newly established method is simpler and applies for quantitative analysis. Ginsenosides developed by acetonitrile-water-acetic acid solvent system on a PES membrane were directly treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a ginsenoside-BSA conjugate on a PES membrane. Anti-G-Rb1 monoclonal antibody (MAb) was bound, and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted to the enzyme and gave staining. The stained membrane was scanned, and spots were analyzed quantitatively using NIH Image software. At least 62.5 ng of G-Rb1, G-Rc, and G-Rd were clearly detectable individually. Three ginsenosides can be analyzed quantitatively between 0.125 and 2.0 microg.

Antibodies, Monoclonal↗