Infusion of gliotoxins or a gap junction blocker in the prelimbic cortex increases alcohol preference in Wistar rats.

Postmortem research has revealed that there is a lower density of glial cells in regions of the prefrontal cortex (PFC) of uncomplicated alcoholics when compared with control subjects. Impairment of astrocyte function in the PFC may contribute to malfunction in circuits involved in emotion- and reward-related subcortical centers, heavily connected with the PFC and directly involved in the pathophysiology of addictive behaviours. The hypothesis was tested that infusion of gliotoxins known to injure astrocytes or of a gap junction blocker into the prelimbic area of the rat PFC results in increased preference for ethanol in rats exposed to free choice between water and 10% ethanol. Fluorocitric acid, L-alpha-aminoadipic acid (AAD) or the gap junction blocker 18-alpha-glycyrrhetinic acid (AGA) were bilaterally infused once into the rat prelimbic cortex and alcohol preference (ratio of 10% ethanol consumed to total liquid ingested) was measured before and after infusion. Infusion of AAD or AGA dissolved in their vehicles, but not of their vehicles alone, resulted in significant transient increase of preference for 10% ethanol. The present data suggest that impaired integrity of glial cells or the gap junctional communication between them in the rat PFC may contribute to changes in ethanol preference.

A novel analytical ELISA-based methodology for pharmacologically active saikosaponins.

A competitive enzyme-linked immunosorbent assay (ELISA) for saikosaponins was established using monoclonal antibody (MAb) 3G10. Hybridoma 3G10 prepared by fusing splenocytes immunized with saikosaponin a-BSA (SSa-BSA) conjugate and a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-U1, secreted monoclonal antibodies with wide cross-reactivity to saikosaponins including saikosaponin b(2) (SSb(2)), c (SSc) and d (SSd), which are stereo and/or regio isomers of SSa. The method, at an effective measuring range of 0.6 mug /ml to 2.3 mug/ml of SSa, successfully detected total saikosaponins in Bupleuri radix and Kampo medicines prescribed with Bupleuri radix. Good correlation between ELISA and HPLC analyses of total saikosaponin in a crude extract of Bupleuri radix was obtained after hydrolysis of acyl saikosaponins by treatment with a mild alkaline solution.

Production of monoclonal antibody against ginkgolic acids in Ginkgo biloba Linn.

A competitive enzyme-linked immunosorbent assay (ELISA) for ginkgolic acids (GAs) was developed using monoclonal antibody (MAb) 9F raised against 6-(13-formylheptyl) salicylic acid covalently coupled to bovine serum albumin (BSA). ELISA, at an effective measuring range of 300 ng/ml-1 microgram/ml of GA15:1, was successful in detecting GAs content in ginkgo leaves and standardized extracts due to the lack of cross-reactivity against various related compounds. The sensitive and simple immunoassay developed in this study was validated to be specific for the quantitative determination of total GAs content in ginkgo crude drugs with no interference from the sample matrix. The analytical recovery of spiked GA15:1 was 103% in a concentration range between 10 and 40 mg/g dry weight of ginkgo leaves.

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants.

We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

Pleomorphic adenoma in the lung.

Pleomorphic adenoma arising from right middle lobe bronchus is reported in an 18-year-old female. Right middle lobectomy was performed and the histologic pattern of the resected tumor was indistinguishable from that commonly described for pleomorphic adenoma of the salivary glands. Mitotic figures were not present. There has been no recurrence two years following surgery.

Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

Double staining of ginsenosides by Western blotting using anti-ginsenoside Rb1 and Rg1 monoclonal antibodies.

Ginsenosides separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane was treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a ginsenoside-BSA conjugate on a PVDF membrane. The blotted bands were stained with anti-ginsenoside Rg1 and Rb1 monoclonal antibodies (MAbs). The newly established double staining with the Western blotting methods was applied for the determination of ginsenosides possessing protopanaxadiol or protopanaxatriol and the number of sugar depending on the stained color and their Rf values in the Panax plants.

Production of monoclonal antibodies against a major purgative component, sennoside B, their characterization and use in ELISA.

For immunization, sennoside B was conjugated with bovine serum albumin. The hapten density in the antigen conjugate was determined to be 3 mol mol(-1) protein by matrix-assisted laser desorption-ionization TOF mass spectrometry. A hybridoma secreting monoclonal antibody against sennoside B was produced by fusing splenocytes from mouse immunized with the sennoside B conjugate and mouse myeloma cells. Weak cross-reactivities occurred with sennoside A which is a stereochemical isomer, and a monomer of sennoside B, rhein, but no cross-reactivity was observed with other related anthraquinones and phenolics. The range of the assay extended from 0.5 ng ml(-1) to 15 ng ml(-1) of sennoside B, and good correlation between ELISA and HPLC methods was obtained when crude extracts of rhubarb were analyzed.

Morphine metabolism in the opium poppy and its possible physiological function. Biochemical characterization of the morphine metabolite, bismorphine.

We identified a novel metabolic system of morphine in the opium poppy (Papaver somniferum L.). In response to stress, morphine is quickly metabolized to bismorphine consisting of two morphine units, followed by accumulation in the cell wall. This bismorphine binds predominantly to pectins, which possess high galacturonic acid residue contents, through ionical bonds. Our newly developed method using artificial polysaccharides demonstrated that bismorphine bridges are formed between the two amino groups of bismorphine and the carboxyl groups of galacturonic acid residues, resulting in cross-linking of galacturonic acid-containing polysaccharides to each other. The ability of bismorphine to cross-link pectins is much higher than that of Ca2+, which also acts as a cross-linker of these polysaccharides. Furthermore, we confirmed that cross-linking of pectins through bismorphine bridges leads to resistance against hydrolysis by pectinases. These results indicated that production of bismorphine is a defense response of the opium poppy. Bismorphine formation is catalyzed by anionic peroxidase that pre-exists in the capsules and leaves of opium poppies. The constitutive presence of morphine, together with bismorphine-forming peroxidase, enables the opium poppy to rapidly induce the defense system.

Endogenous cannabinoid, 2-arachidonoylglycerol, attenuates naloxone-precipitated withdrawal signs in morphine-dependent mice.

In the present study, we examined the effects of endogenous ligand 2-arachidonoylglycerol (2-AG) on naloxone-precipitated withdrawal in morphine-dependent mice, in comparison with that of two cannabinoid agonists, an ingredient of Cannabis sativa Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and the synthetic cannabinoid CB1 receptor agonist HU-210. 2-AG at a dose of 10 microg per mouse (i.c.v.) significantly inhibited both jumping and forepaw tremor as signs of withdrawal following naloxone challenge in morphine-dependent mice. Furthermore, both Delta(8)-THC and HU-210 significantly attenuated these symptoms of withdrawal in morphine-dependent mice. Therefore, it is suggested that inactivation of the endogenous cannabinoid system is related to the induction of withdrawal syndrome in morphine-dependent mice. Moreover, hyperlocomotor activity in morphine-dependent mice was markedly increased by Delta(8)-THC 10 mg/kg, which had no effect in naive mice. This finding suggested that in morphine dependence, upregulation of cannabinoid CB1 receptors occurred. Non-psychoactive CB1 receptor agonists or accelerators of endocannabinoid synthesis may be potential as therapeutic drugs for opiate withdrawal symptoms.

Effects of naturally occurring glucosides, solasodine glucosides, ginsenosides and parishin derivatives on multidrug resistance of lymphoma cells and leukocyte functions.

Solamargine, solasonine, ginsenosides and parishin-related compounds were investigated for their effects on mdr efflux pump of lymphoma cells, and their effects on T cell proliferative assays and cell mediated immune functions, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell activity of human peripheral mononuclear cells. Solamargine and solasonine were the only drugs which inhibited all of the tested immune functions; however, ginsenoside Rc and Rd enhanced T cell proliferative assays and marginally increased the NK cell activity. The majority of the compounds were not able to reverse the multidrug resistance of mouse lymphoma cells. However, ginsenosides Rc, Rd and parishin C were able to moderately reduce the activity of the efflux pump. Parishin, parishin C and crude extract significantly enhanced the ADCC reaction.

Higher yielding isolation of kinsenoside in Anoectochilus and its antihyperliposis effect.

A higher concentration of kinsenoside, 3-(R)-3-beta-D-glucopyranosyloxybutanolide (1), was detected in the crude drug Anoectochilusformosanus, and A. koshunensis by HPLC analysis. A methylation reaction occurred to give methyl ester (4) when the lactone ring of 1 was cleaved by silica gel catalysis using methanol containing solvent used in the purification step resulting in difficulty to purify 1. To avoid the cleavage reaction, a reversed-phase or silica gel column without methanol was used to give a high yield of 1. In an anti-hyperliposis assay using high-fat diet rats, 1 significantly reduced the weights of body and liver, and also decreased the triglyceride level in the liver compared to those of control rats. On the other hand, the epimer of 1, 3-(S)-3-beta-glucopyranosy-loxybutanolide (2), trivially named goodyeroside A, which was isolated from Goodyera species, had no effect for anti-hyperliposis. In aurothioglucose-induced obese mouse, 1 suppressed the body and liver weight increase, significantly ameliorated the triglyceride level in the liver, and also reduced the deposition of uterine fat-pads.

Behavioral suppression induced by cannabinoids is due to activation of the arachidonic acid cascade in rats.

Tetrahydrocannabinol (THC) is the principle psychoactive ingredient of marijuana and produces various psychoactive effects through the brain cannabinoid (CB1) receptor. The CB1 receptor belongs to the seven-transmembrane domain family of G-protein-coupled receptors and is involved in the arachidonic acid cascade in the brain. Few reports have attempted to clarify the functional role of endogenous cannabinoid and the arachidonic acid cascade through the CB1 receptor using a behavioral paradigm. Therefore, in this study, we clarified the mechanism of cannabinoid-induced suppression of lever pressing in rats, focusing on the arachidonic acid cascade as a novel second messenger of CB1 receptor. Delta(8)-THC and the potent synthetic CB1 receptor agonist HU-210 dose-dependently inhibited lever-pressing performance. The Delta(8)-THC-induced suppression was significantly antagonized by the cyclooxygenase (COX) inhibitors diclofenac (32 mg/kg, i.p.), aspirin (10 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.). The suppressive effect of HU-210 was also significantly antagonized by 32 mg/kg diclofenac. Prostaglandin E(2) (3.2 microg/rat, i.c.v.), the final product of the arachidonic acid cascade, significantly inhibited lever pressing similar to Delta(8)-THC and HU-210. In conclusion, we found that suppression of lever-pressing behavior induced by cannabinoids was mediated through activation of the arachidonic acid cascade via the CB1 receptor. Therefore, it is possible that the psychoactive effects of cannabinoid are due to an increase in the formation of PGE(2) in the brain.

Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid.

Hybridomas secreting a monoclonal antibody against glycyrrhizin were produced by fusing splenocytes from a mouse immunized against a glycyrrhizin-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very weak cross-reaction with glycyrrhetinic acid monoglucuronide and glycyrrhetic acid occurred. An enzyme-linked immunosorbent assay (ELISA) that had an effective measuring range of 20 -200 ng/mL of glycyrrhizin was established using this monoclonal antibody. In addition, a method named eastern blotting for the detection of glycyrrhizin was investigated. In this method, we developed a new way to separate the glycyrrhizin molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups on the protein and covalently bound to the adsorbent membrane. The monoclonal antibody bound to the aglycone part of the glycyrrhizin molecule for immunostaining. This method was validated by immunocytolocalization of glycyrrhizin in fresh Glycyrrhiza root.

Inhibition of P-glycoprotein by orange juice components, polymethoxyflavones in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells.

We investigated the effects of the ethyl acetate extract of grapefruit juice (GFJ), that of orange juice (OJ) and their components on the uptake of [(3)H]vincristine into adriamycin-resistant human myelogenous leukemia cells. Its uptake was increased by the extracts of GFJ and OJ up to 7- and 3-fold, respectively, as well as verapamil and cyclosporin A. OJ components, i.e. 3,3',4',5,6,7,8-heptamethoxyflavone, nobiletin and tangeretin, also increased the uptake of [(3)H]vincristine in a concentration-dependent manner. Although GFJ components, dihydroxybergamottin and bergamottin, significantly increased the uptake, their potencies were considerably weaker than those of OJ components. These data suggest that OJ components inhibit P-gp-mediated efflux of [(3)H]vincristine, resulting in the intracellular accumulation of chemotherapeutic drugs. These components may become candidates of multi-drug resistance reversing agents in cancer chemotherapy.

Inhaled flow and handling of fluticasone diskhaler by asthmatic patients.

We investigated the inhaled flow and handling of a Fluticasone Diskhaler (FDH) by patients familiar with the beclomethasone dipropionate inhaler (BDI), a metered dose inhaler. Before the FDH was introduced in our hospital, 174 patients were instructed in the use of the Diskhaler and measured the flow of Diskhaler inhalation. Three months after the introduction of the FDH, approximately 95 patients were using them. During their regular visit to the hospital, we checked the patients' handling of FDH and the flow of FDH inhalation (n=81). It was found that only 22% of the patients correctly handled the FDH. The major erros concerned breath-holding and disk rotation after use, but 9.9% of the patients handled the inhaler with serious error, e.g., incomplete puncturing of the blister. The mean FDH flow was 86.5 L/min, which was significantly higher than that recorded at the first FDH trial (69.1 L/min). In 9.9% of the patients, the inhaled flow was inappropriately low (<50 L/min), in 29.6% of the patients, it was unnecessarily high (>100 L/min). In conclusion, handling the FDH is easy for patients who are already familiar with the BDI. However, in 40% of the patients, the inhaled flow rate was not sufficient.

Glycosidic constituents from in vitro Anoectochilus formosanus.

The glycosidic constituents of whole plants of Anoectochilus formosanus propagated by tissue culture were investigated. A new compound, 2-(beta-D-glucopyranosyloxymethyl)-5-hydroxymethylfuran, along with the known compounds, 3-(R)-3-beta-D-glucopyranosyloxybutanolide (kinsenoside), 3-(R)-3-beta-D-glucopyranosyloxy-4-hydroxybutanoic acid, 1-O-isopropyl-beta-D-glucopyranoside, (R)-(+)-3,4-dihydroxy-butanoic acid y-lactone, 4-(beta-D-glucopyranosyloxy)benzyl alcohol, (6R,9S)-9-hydroxy-megastigma-4,7-dien-3-one-9-O-beta-glucopy ranoside, and corchoionoside C were isolated.

[Pharmacognosical study on secondary metabolites].

Clonal micropropagation on various medicinal plants was set up resulting in the regenerated plants which possessed a homogeneous quality. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization of mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigene-BSA conjugate with mouse myeloma cells. Competitive enzyme-linked immunosorbent assay (ELISA) using MAb was set up as a high sensitive, specific and reproducible qualitative method. A method of determination for ginsenosides by using a unique western blotting was established. Immunoaffinity column chromatography using an anti-ginsenoside Rb1MAb has made possible a single-step separation of ginsenoside Rb1 from a crude ginseng extract. Single chain Fv gene of anti-forskolin MAb was prepared from mRNA of hybridoma secreting anti-forskolin MAb and cloned. Gene was constructed into a pET-28a(+) vector producing a scFv protein. Modeling of forskolin and scFV was investigated. THCA synthase was purified from the homogenate of Cannabis sativa leaves on successive column chromatographies. THCA synthase was confirmed to be homogeneity having 75 kDa. To obtain the corresponding cDNA clone of THCA synthase, a set of degenerate promers was constructed based on N-terinal and internal amino acid sequences of THCA synthase. The 5' and 3' ends of cDNA were amplified by RACE. A full sequencing has been determined to be corded a polypeptide having 545 amino acid residues. The cDNA clone was expressed in yeast system via PUC19 vector resulting in THCA synthase activity.

Applications of ELISA, western blotting and immunoaffinity concentration for survey of ginsenosides in crude drugs of Panax species and traditional Chinese herbal medicines.

A combination of ELISA, Western blotting and immunoaffinity concentration using an anti-ginsenoside Rb1 monoclonal antibody was applied for qualitative and quantitative surveys of ginsenoside Rb1 and related ginsenosides in roots and traditional Chinese herbal medicines. To improve the low correlation between ELISA and HPLC analysis, the crude extract of roots was immunoaffinity concentrated to make evident the effect of malonyl ginsenoside Rb1. Immunoaffinity column chromatography also concentrated an unknown ginsenoside which had the same cross-reaction with ginsenoside Rb1.

Effect of furanocoumarin derivatives in grapefruit juice on the uptake of vinblastine by Caco-2 cells and on the activity of cytochrome P450 3A4.

1. The presence of inhibitors of drug efflux transporters, such as P-glycoprotein (P-gp), in grapefruit juice (GFJ) was confirmed based on the uptake of [(3)H]-vinblastine (VBL) by Caco-2 cells. 2. The uptake of [(3)H]-VBL by Caco-2 cells was significantly increased by the ethyl acetate extract of GFJ as well as by cyclosporin A. The extract was separated on a Cosmosil column and the eluate with 60% methanol increased [(3)H]-VBL uptake, while the activity to inhibit CYP3A4 was greatest in the 70 and 80% eluates. 3. These results show that the major inhibitor of efflux transport of VBL is different from that of CYP3A4. 4. Further separation of the 60% methanol eluate afforded dihydroxybergamottin (DHBG). Both ethyl acetate extract of GFJ and DHBG increased steady-state [(3)H]-VBL uptake by LLC-GA5-COL300 cells. Besides DHBG, other furanocoumarins contained in GFJ, such as bergamottin, FC726, bergaptol and bergapten, increased the steady-state uptake of [(3)H]-VBL by Caco-2 cells. 5. The order of inhibitory potency of these compounds was FC726>DHBG>bergamottin>bergapten>bergaptol . While, the IC(50) values for inhibition of CYP3A4 were 0.075, 0.45, 1.0, 1.0 and >20 microM, respectively. Bergaptol specifically inhibited VBL efflux. 6. DHBG was thus identified as a candidate for inhibitors of VBL transport, together with other furanocoumarins. Moreover, partly involvement of the P-gp inhibition was suggested. 7. Therefore, the inhibition of efflux transport of drugs as well as of drug metabolism by CYP3A4 could be an important cause of drug-GFJ interaction.