Yokukansan inhibits morphine tolerance and physical dependence in mice: the role of α2A-adrenoceptor.

Yokukansan (YKS) is a traditional Japanese medicine consisting of seven medicinal herbs that is used for the treatment of neurosis, insomnia, and the behavioral/psychological symptoms of dementia. This study examined the effects of YKS on morphine tolerance and physical dependence in mice. Daily oral administration of YKS (0.5 or 1.0 g/kg) for 3 weeks significantly attenuated morphine tolerance and naloxone-precipitated morphine withdrawal signs (jumps and body weight loss) without affecting the analgesic effect of morphine. The inhibitory effect of YKS on withdrawal jumps in morphine-dependent mice was blocked by a single pretreatment with an α(2)-adrenoceptor antagonist, yohimbine, but not by an α(1)-adrenoceptor antagonist, prazosin. A similar inhibitory effect on withdrawal jumps was observed by repeated administration of yohimbine. The membrane expression of α(2A)-adrenoceptors in the pons/medulla was decreased in morphine withdrawn animals; this reduction was prevented by repeated administration of YKS or yohimbine. Competitive radioligand and [(35)S]guanosine-5'-O-(3-thiotriphosphate) binding assays revealed that YKS and its constituent herbs, Glycyrrhiza (GR) and Uncaria hook (UH), had specific binding affinity for and antagonist activity against the α(2A)-adrenoceptor. Certain chemical constituents, including GR -derived glycyrrhizin and its metabolite, 18ß-glycyrrhetinic acid, and UH-derived geissoschizine methyl ether (GME), shared such activities. Repeated administration of GR, UH, glycyrrhizin or GME significantly inhibited morphine withdrawal signs. These results suggest that YKS and its active constituents inhibit morphine tolerance and physical dependence, and that the latter is due at least in part to the prevention of the decreased membrane expression of the α(2A)-adrenoceptor in the brainstem by its prolonged blockade.

Concomitant use of enteral nutrition therapy is associated with sustained response to infliximab in patients with Crohn's disease.

BACKGROUND/OBJECTIVES: A significant proportion of Crohn's disease (CD) patients receiving infliximab (IFX) maintenance therapy show loss of responsiveness despite a good initial response. The factors other than immunomodulators that prevent IFX dose escalation have yet to be fully elucidated. This study was performed to identify clinical factors or concomitant therapies associated with sustained response to IFX. SUBJECTS/METHODS: Seventy-four consecutive CD patients who had successful IFX induction therapy between 2002 and 2010 underwent IFX maintenance therapy. Patients showing loss of response to IFX were treated with IFX intensification therapy. Factors involved in the sustained response to IFX were investigated retrospectively. RESULTS: After a median follow-up of 85 weeks, loss of response to IFX was observed in 30 (40.5%) cases. On logistic regression analysis, concomitant use of enteral nutrition (EN) therapy (elemental and/or polymeric formulas) was identified as an independent factor associated with sustained response to IFX. Receiver operating characteristic curve analysis indicated a cutoff value of 600 kcal/day. We divided the patients into the 'EN group' (≥ 600 kcal/day) and 'control group' (<600 kcal/day). The cumulative number of loss of response was significantly lower in the EN group (odds ratio: 0.23, P = 0.0043). Kaplan-Meier analysis confirmed the significantly lower rate of loss of response in the EN group (P = 0.013). Multivariate hazard ratio was 0.37 (P = 0.025). Type of EN formula did not affect the results. CONCLUSIONS: Concomitant use of EN ≥ 600 kcal/day is likely to yield a sustained response to IFX in CD patients.

Discovery of potent and selective histamine H3 receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold.

A novel series of potent histamine H(3) receptor inverse agonists based on the 3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one scaffold has been discovered. Several compounds display high selectivity over other histamine receptor subtypes and have favorable physicochemical properties, low potential for CYP450 enzyme inhibition and high metabolic stability in microsomal preparations. (R)-2-Cyclopropylmethyl-8-(1-isopropyl-piperidin-4-yloxy)-3-methyl-3,4-dihydro-2H-pyrazino[1,2-a]indol-1-one (8t) showed good in vivo efficacy after per os application in an acute rat dipsogenia model of water intake.

Long-term infusion of brain-derived neurotrophic factor reduces food intake and body weight via a corticotrophin-releasing hormone pathway in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) has been implicated in learning, depression and energy metabolism. However, the neuronal mechanisms underlying the effects of BDNF on energy metabolism remain unclear. The present study aimed to elucidate the neuronal pathways by which BDNF controls feeding behaviour and energy balance. Using an osmotic mini-pump, BDNF or control artificial cerebrospinal fluid was infused i.c.v. at the lateral ventricle or into the paraventricular nucleus of the hypothalamus (PVN) for 12 days. Intracerebroventricular BDNF up-regulated mRNA expression of corticotrophin-releasing hormone (CRH) and urocortin in the PVN. TrkB, the receptor for BDNF, was expressed in the PVN neurones, including those containing CRH. Both i.c.v. and intra-PVN-administered BDNF decreased food intake and body weight. These effects of BDNF on food intake and body weight were counteracted by the co-administration of alpha-helical-CRH, an antagonist for the CRH and urocortin receptors CRH-R1/R2, and partly attenuated by a selective antagonist for CRH-R2 but not CRH-R1. Intracerebroventricular BDNF also decreased the subcutaneous and visceral fat mass, adipocyte size and serum triglyceride levels, which were all attenuated by alpha-helical-CRH. Furthermore, BDNF decreased the respiratory quotient and raised rectal temperature, which were counteracted by alpha-helical-CRH. These results indicate that the CRH-urocortin-CRH-R2 pathway in the PVN and connected areas mediates the long-term effects of BDNF to depress feeding and promote lipolysis.

Characterizing kinetics of transport and transformation of selenium in water-sediment microcosm free from selenium contamination using a simple mathematical model.

This study developed a seven-compartment model for predicting the fate of selenium (Se) in an aquatic environment containing a water-sediment boundary. Speciation of Se in water-sediment microcosms under microaerobic conditions was measured to evaluate first-order kinetics of Se transportation and transformation. The microcosm consisted of a 10-ml solution containing 1mM soluble Se as selenate (Se6+) or selenite (Se4+) and 8 g wet sediment that was free from Se contamination, sampled from the Senri, Yamato, or Yodo Rivers in Osaka, Japan. Stepwise reaction coefficients describing transportation and transformation were determined using an inverse method on this model which includes: selenate (Se(W)6+) and selenite (Se(W)4+) in ponded water; selenate (Se(S)6+) and selenite (Se(S)4+), elemental Se (Se0), organic Se (Se2-) in sediment; and gaseous Se (DMSe). During this 1-month experiment, soluble Se was transported from ponded water to the sediment and Se was transformed sequentially to other Se species through biochemical reactions. Experimental and kinetic analyses indicated quantitatively that the Yamato River microcosm, with its high organic matter content, had a high adsorption rate of soluble Se. The Yodo River microcosm had a low adsorption rate for Se6+ and a low Se reduction rate. The Senri River microcosm had an apparent high volatilization rate of DMSe. The model developed in this study is extremely useful for predicting fate of Se in aquatic environment in the field.

Isolation and characterization of psychrophilic yeasts producing cold-adapted pectinolytic enzymes.

AIMS: The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold-active pectinolytic enzymes produced by the isolated psychrophilic yeasts. METHODS AND RESULTS: Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic-psychrophilic yeast (PPY) strains PPY-3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY-3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY-5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5 degrees C, and showed the activities of several cold-active pectinolytic enzymes. CONCLUSION: The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible that the cold-active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5 degrees C.

Recent developments in clinical trials for bronchial asthma.

Bronchial asthma is a chronic inflammatory disorder of the airways. Currently available antiinflammatory treatments, represented by inhaled corticosteroids (ICS), are highly effective in controlling symptoms in the majority of patients, but their potential side effects have led to the use of adjunctive or alternative therapies and to the development of new therapies. Most of these new agents are aimed at inhibiting various components of allergic inflammation, with better safety profiles than ICS. They include inhibitors of phosphodiesterase 4, cytokine modulators, chemokine receptor antagonists and antisense oligonucleotides.

Anti-obesity and anti-diabetic effects of brain-derived neurotrophic factor in rodent models of leptin resistance.

OBJECTIVE: Obesity in rodents and humans is mostly associated with elevated plasma leptin concentrations, suggesting a new pathological concept of 'leptin resistance'. We have demonstrated that brain-derived neurotrophic factor (BDNF) can improve obesity and diabetes of C57BL/KsJ db/db (db/db) mice. In this study, we investigated whether or not BDNF is effective in two different models of leptin resistance, an acquired model and a genetic model. DESIGN: C57BL/6J mice rendered obese by consumption of a high-fat diet (diet-induced obesity (DIO) mice) were used as an acquired model and lethal yellow agouti mice (KKA(y) mice) as a genetic model of leptin resistance. Food intake and glucose metabolism were studied after acute or repetitive administration of BDNF. RESULTS: Intraperitoneal administration of BDNF (10 mg/kg, twice/day) significantly reduced cumulative food intake of DIO and KKA(y) mice, whereas they were unresponsive to leptin administration. Repetitive subcutaneous administration of BDNF (10 mg/kg daily for 6 days) reduced food intake and improved impaired glucose tolerance in DIO mice. Pair feeding of vehicle-treated DIO mice with the same amount of chow consumed by the BDNF-treated group did not improve the impaired glucose homeostasis, indicating that the antidiabetic effect is not due to decreased food intake. We also observed that BDNF is effective in improving obesity and diabetes of KKA(y) mice. CONCLUSION: This study demonstrated antiobesity and antidiabetic effects of BDNF in two different models of leptin resistance, thereby suggesting the therapeutic potential of BDNF in the treatment of leptin-resistant obesity and diabetes.

The effects of Coptidis Rhizoma extract on a hypercholesterolemic animal model.

The serum cholesterol (total, free, esterified, low density lipoprotein (LDL) and oxidized LDL) levels of rats fed a diet containing, by weight, 1% cholesterol and 0.5% cholic acid increased, as compared with those of rats fed a normal diet. The levels, especially of total cholesterol, LDL and oxidized LDL, were reduced significantly in a dose-dependent manner, in rats given Coptidis Rhizoma extract orally at doses of 50 and 100 mg/kg body wt./day for 30 days. These results indicate that Coptidis Rhizoma extract is effective in reducing the pathological damage caused by hypercholesterolemia, through lowering of serum cholesterol levels. In addition, Coptidis Rhizoma extract reduced the level of liver cholesterol, but it did not reduce that of fecal cholesterol, suggesting that the cholesterol level-lowering effect resulted from the reduction of cholesterol synthesis, not the enhancement of its excretion. Furthermore, the serum thiobarbituric acid-reactive substance level decreased after oral administration of Coptidis Rhizoma extract, indicating that Coptidis Rhizoma could prevent hypercholesterolemic disease through reducing lipid peroxidation. This study demonstrates that Coptidis Rhizoma may be a useful therapy for hypercholesterolemia through reducing oxidative stress and cholesterol levels.

Direct scavenging of nitric oxide and superoxide by green tea.

In the present study, we investigated the free radical scavenging effects of green tea extract and green tea tannin mixture and its components using a nitric oxide (NO) and superoxide (O(2)(-)) generating system in vitro. Green tea extract showed direct scavenging activity against NO and O(2)(-) and green tea tannin mixture, at the same concentration, showed high scavenging activity. Comparison of the activities of seven pure compounds isolated from green tea tannin mixture showed that (-)-epigallocatechin 3-O-gallate (EGCg), (-)-gallocatechin 3-O-gallate (GCg) and (-)-epicatechin 3-O-gallate (ECg) had higher scavenging activities than (-)-epigallocatechin (EGC), (+)-gallocatechin (GC), (-)-epicatechin (EC) and (+)-catechin (C), showing the importance of the structure of flavan-3-ol linked to gallic acid for this activity. Among the gallate-free tannins, EGC and GC were more effective O(2)(-) scavengers than EC and C, indicating the O-trihydroxy structure in the B ring is an important determinant of such activity. However, this structure did not affect the NO scavenging activity. These findings confirm that green tea tannin has excellent antioxidant properties, which may be involved in the beneficial effect of this compound.

Introduction of recirculatory analysis into portal and systemic concentration difference method.

Recirculatory analysis was introduced into the portal and systemic concentration difference method with double dosing (PS-DD method), which is an evaluation system for the local intestinal and hepatic first-pass effect. 5-Fluorouracil (5-FU) and cephalexin (CEX) were selected as model drugs. A new recirculatory system was constructed to predict the time courses of a drug concentration in the systemic and portal bloods. Bioavailability (F), local absorption ratio (Fa), hepatic recovery ratio (FH), and local mean absorption time (ta) estimated by recirculatory analysis were close to those calculated by moment analysis with numerical integration. Using recirculatory analysis, the sampling period was considerably shortened and the sampling number was also reduced, which demonstrates that recirculatory analysis is useful in PS-DD method.

Effect of oral treatment of Perilla frutescens and its constituents on type-I allergy in mice.

Perilla frutescens Britton (perilla, Labiatae) is a medicinal herb prescribed in Saiboku-to [Japanese letters: see text], which is a Kampo formula effective for allergic diseases such as bronchial asthma. The present study was conducted to evaluate the anti-allergic effect of orally administered perilla decoction and to identify the active constituents using mice ear-passive cutaneous anaphylaxis (PCA)-reaction, which is one of the animal models for type I allergy. Perilla decoction significantly suppressed PCA-reaction, and the inhibition % at the dose of 500 mg/kg was 43%. The perilla decoction contains 5.3% of luteolin 7-O-[beta-glucuronosyl(2-->1)beta-glucuronide], 1.6% of apigenin 7-O-[beta-glucuronosyl(2-->1)beta-glucuronide], 0.49% of scutellarin, and 2.5% of rosmarinic acid (weight of compound/dried weight of perilla decoction %), respectively. When these constituents were orally administered to mice at the dose equivalent to 500 mg/kg of perilla decoction, rosmarinic acid and apigenin 7-O-[beta-glucuronosyl(2-->1)beta-glucuronide] significantly suppressed PCA-reaction, and their inhibition % was 41% (p<0.01) and 32% (p<0.05), respectively. Since the inhibition % or perilla decoction and rosmarinic acid were nearly equal, the anti-allergic effect of perilla decoction depends primarily on rosmarinic acid. The standard Saiboku-to decoction contained 0.013% of rosmarinic acid, which was too low to exhibit anti-allergic activity in a daily dose of Saiboku-to in adults, suggesting that perilla would be prescribed in Saiboku-to to exhibit other pharmacological effects than its anti-allergic activity, such as a sedative.

A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species.

Many flowering plants have evolved self-incompatibility (SI) systems to prevent inbreeding. In the Brassicaceae, SI is genetically controlled by a single polymorphic locus, termed the S-locus. Pollen rejection occurs when stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cys-rich (SP11/SCR). SRK encodes a polymorphic membrane-spanning protein kinase, which is the sole female determinant of the S-haplotype specificity. SP11/SCR encodes a highly polymorphic Cys-rich small basic protein specifically expressed in the anther tapetum and in pollen. In cauliflower (B. oleracea), the gain-of-function approach has demonstrated that an allele of SP11/SCR encodes the male determinant of S-specificity. Here we examined the function of two alleles of SP11/SCR of B. rapa by the same approach and further established that SP11/SCR is the sole male determinant of SI in the genus Brassica sp. Our results also suggested that the 522-bp 5'-upstream region of the S9-SP11 gene used to drive the transgene contained all the regulatory elements required for the unique sporophytic/gametophytic expression observed for the native SP11 gene. Promoter deletion analyses suggested that the highly conserved 192-bp upstream region was sufficient for driving this unique expression. Furthermore, immunohistochemical analyses revealed that the protein product of the SP11 transgene was present in the tapetum and pollen, and that in pollen of late developmental stages, the SP11 protein was mainly localized in the pollen coat, a finding consistent with its expected biological role.

A methylotrophic pathway participates in pectin utilization by Candida boidinii.

The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

Cystine protects cochlear outer hair cells against glutamate toxicity.

We previously reported that long-term exposure to glutamate (Glu) induced death of cochlear outer hair cells (OHCs). However, the mechanisms of OHC death induced by Glu were unclear. In the central nervous system, Glu is known to interfere with a cystine-Glu antiporter, leading to a decrease in cystine uptake and reducing the intracellular glutathione level. We therefore investigated the effect of cystine supplementation on degeneration of OHCs caused by long-term exposure to Glu. Supplementation of cystine significantly decreased the number of dying OHCs. These findings suggest that a cystine-Glu interaction may be involved in the mechanism of OHC degeneration caused by Glu.

The traditional Chinese medicine banxia houpo tang improves swallowing reflex.

A marked depression of swallowing reflex has been found in patients with aspiration pneumonia. We have examined the effects of Banxia Houpo Tang (BHT, Hange Koboku-To in Japanese), on swallowing reflex among the elderly. Thirty-two patients, mean age 74.2 +/- 1.7 years who had at least one episode of aspiration pneumonia, were divided into two groups. Twenty patients took BHT extracts of 7.5 g per day for four weeks, and the other 12 patients took a placebo. The swallowing reflex was measured by a bolus injection of 1 ml of distilled water into the pharynx through a nasal catheter. The reflex was evaluated by the latency time of response, which was the time from the injection to the onset of swallowing. The latency of response decreased significantly from 11.6 +/- 3.0 sec to 2.6 +/- 0.4 sec in the group treated with BHT (p < 0.01), while in the other group with placebo it was from 11.0 +/- 4.0 to 10.8 +/- 3.6 (p > 0.5). Depletion of substance P in the pharynx causes impairments of the swallowing reflex. Substance P in the saliva of treated patients increased from 9.2 +/- 2.5 fmol/ml to 15.0 +/- 2.2 fmol/ml after BHT treatment (p < 0.01), while levels were 8.0 +/- 4.0 fmol/ml before and 7.1 +/- 3.1 fmol/ml after among the placebo group (no significant difference). We suggest that BHT improves the impaired swallowing reflex and may help to prevent aspiration pneumonia in the elderly.

Intracerebroventricular administration of nocistatin reduces inflammatory hyperalgesia in rats.

Nocistatin is a biologically active peptide derived from prepronociceptin, and its intrathecal administration has been reported to reduce nociceptin- or prostaglandin E2-induced hyperalgesia and allodynia in mice. In this study, we investigated the effects of intracerebroventricular (i.c.v.) administration of nocistatin on the inflammatory hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin in the rat paw-pressure test. Intracerebroventricular administration of nocistatin (0.5-50 pmol/rat) dose-dependently reduced carrageenan/kaolin-induced hyperalgesia, which peaked at 15-30 m. However, i.c.v. administration of nocistatin (50 pmol/rat) had no effect on the nociceptive threshold of non-inflamed rats. These results indicate that nocistatin has anti-hyperalgesic effects on the inflammatory hyperalgesia induced by carrageenan/kaolin at the supraspinal level.

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene.

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.

Effects of green tea tannin on cisplatin-induced nephropathy in LLC-PK1 cells and rats.

A study was conducted to clarify whether green tea tannin ameliorated cisplatin-induced renal injury in terms of lactate dehydrogenase and malondialdehyde leakage from a renal epithelial cell line, swine-derived LLC-PK1 cells in culture. Green tea tannin was shown to suppress the cytotoxicity of cisplatin, the suppressive effect increasing with the dose of green tea tannin. The effect of cisplatin was then investigated in rats given green tea tannin for 40 days before cisplatin administration and in control rats given no green tea tannin. In control rats, blood, urinary and renal parameters and the activities of antioxidative enzymes in renal tissue deviated from the normal range, indicating dysfunction of the kidneys. In contrast, rats given green tea tannin showed decreased blood levels of urea nitrogen and creatinine, and decreased urinary levels of protein and glucose, reflecting less damage to the kidney. In this group, the activity of catalase in the renal tissue was increased, while the level of malondialdehyde was decreased, suggesting the involvement of radicals in the normalizing of kidney function. Based on the evidence available it appeared that green tea tannin eliminated oxidative stress and was beneficial to renal function.

Dose-dependent response of vestibular hair cells of guinea pigs following streptomycin ototoxiation.

Although the involvement of apoptosis has been suggested in the loss of vestibular hair cells due to aminoglycosides, dose-dependent effects of aminoglycosides have not been determined. We therefore examined dose-dependent effects of streptomycin on the degeneration of hair cells of guinea pig ampullar cristae using TUNEL stain and Hoechst nuclear stain. Streptomycin induced apoptosis of hair cells in a dose-dependent manner. Even following high-dose applications, most of the affected cells showed apoptotic features. Apoptosis may therefore play a predominant role in the deletion of vestibular hair cells affected by aminoglycosides.