Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation.

The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund's adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (V(h)=-60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 mum), voltage-dependent transient K(+) current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltage-dependent transient K(+) current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at two-fold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund's adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient K(+) current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder.

Role of capsaicin-sensitive primary afferent inputs from the masseter muscle in the C1 spinal neurons responding to tooth-pulp stimulation in rats.

The aim of the present study was to demonstrate the convergence of inputs from masseter muscle (MM) and tooth pulp (TP) onto C1 spinal neurons and to determine whether the afferent fibers express the functional vanilloid receptor (VR1). Extracellular single-unit recordings were made from 61 C1 units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram signal in pentobarbital anesthetized rats. Eighty-four percent of C1 neurons responding to TP stimulation also responded to the ipsilateral MM stimulation. Of these neurons, 61% were considered to be afferent inputs from Adelta-fibers and the remaining units (39%) were C-fibers, based on calculation of the nerve conduction velocity. Intramuscular injection of capsaicin (0.05 and 0.1%) produced a reduction in a MM-induced C1 neuronal activity in a dose-dependent manner and this effect was antagonized by pretreatment with an antagonist of VR1, capsazepine. Some of these units were also excited by noxious heat stimulation (> 43 degrees C). The trigeminal root ganglion (TRG) neurons that innervated the MM were retrogradely labeled with Fluorogold (FG) and the small-diameter FG-labeled TRG neurons expressed the immunoreactivity for VR1. After intramuscular mustard oil injection (noxious chemical stimulation), the C1 neuronal activity induced by both touch and pinch stimuli was enhanced and their receptive field sizes were significantly expanded. These changes were reversed within 15-20 min. These results suggest that there may be the convergence of noxious afferents inputs from the MM and TP afferents on the same C1 neurons in rats, and that the afferent fibers expressing the functional VR1 may contribute to the hyperalgesia and/or referred pain associated with temporomandibular joint disorder.

Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression.

Despite its beneficial role in host defense mechanisms, excessive nitric oxide (NO) production by activated macrophages has been implicated in several inflammatory diseases. To clarify the mechanisms of anti-inflammatory activities of Polygonum tinctorium, we evaluated whether extracts of P. tinctorium could modulate the production of NO by activated macrophages. An AcOEt extract of P. tinctorium markedly inhibited NO synthesis by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages and the macrophage-like cell line RAW 264.7 in a dose-dependent manner. Inhibition of NO synthesis was achieved by reducing inducible NO synthase (iNOS) expression at protein and mRNA levels. However, the AcOEt extract of P. tinctorium failed to inhibit NO synthesis when iNOS was already expressed following stimulation with IFN-gamma and LPS. The AcOEt extract also exhibited inhibitory activity on iNOS expression in human lung epithelial A549 cells stimulated with a combination of IFN-gamma, TNF-alpha and IL-1 beta without affecting the expression of constitutive isoforms of NOS. Furthermore, in vivo injection of the AcOEt extract of P. tinctorium into LPS-treated mice significantly reduced NO synthesis by peritoneal exudate cells under ex vivo conditions. These results suggest that P. tinctorium extract may be a potential therapeutic modulator of NO synthesis in various pathological conditions.

Enzymatic synthesis of N-acetylglucosaminyl-cyclodextrin by the reverse reaction of N-acetylhexosaminidase from jack bean.

Novel heterobranched cyclodextrins (CDs), N-acetylglucosaminyl-cyclodextrins (GlcNAc-CD), were synthesized from a mixture of GlcNAc and alpha, beta, or gamma CD by the reverse reaction of N-acetylhexosaminidase from jack bean. Optimum pH and temperature for the production of GlcNAc-alpha CD by N-acetylhexosaminidase were pH 4.9 and 50-70 degrees C, respectively. The maximum yield of GlcNAc-alpha CD was 17.5% (mol/mol) at the concentration of 1 M GlcNAc and 0.25 M alpha CD. The reverse reaction product, GlcNAc-alpha CD, was separated into two peaks by HPLC analysis on the ODS column. Their structures were identified as 6-O-beta-D-N-acetylglucosaminyl-alpha CD and 2-O-beta-D-N-acetylglucosaminyl-alpha CD by FAB-MS and NMR spectroscopies. N-Acetylhexosaminidase from jack bean also synthesized N-acetylgalactosaminyl-alpha CD from N-acetylgalactosamine and alpha CD.

Changes in c-Fos expression induced by noxious stimulation in the trigeminal spinal nucleus caudalis and C1 spinal neurons of rats after hyperbaric exposure.

The present study aims to test the hypothesis that hyperbaric exposure inhibits nociceptive processing in the trigeminal spinal nucleus caudalis and C1 spinal neurons. We investigated the c-Fos-like immunoreactivity of the brainstem and upper cervical spinal cord (C1 region) following an injection of mustard oil (15 microliters of 20%) into the nasal mucosa of pentobarbital anesthetized rats after exposure to hyperbaric (2-atmospheres, 1 h) and normobaric pressures. After the hyperbaric exposure, the mean number of Fos-immunoreactive neurons in the ipsilateral laminae I-II and III-IV of the trigeminal spinal nucleus caudalis were significantly lower than those in the normobaric condition. Similarly, the mean number of c-Fos positive neurons in the superficial layer (I-II) of the ipsilateral C1 segment were significantly reduced as compared with that in the normobaric condition. When treated with the vehicle alone, no significant difference was detected in the numbers of c-Fos positive neurons in the trigeminal spinal nucleus caudalis and C1 regions between hyperbaric and normobaric conditions. These results suggest that hyperbaric exposure may attenuate nociceptive signals from the area innervated by the trigeminal nerves at the level of both the trigeminal spinal nucleus caudalis and C1 dorsal horn.

[Inhibition of antigen-specific T helper type 2 responses by Perilla frutescens extract].

Perilla frutescens leaf extract (PFE) is known as a natural medicine with anti-allergic activities, although its mechanism of action remains unclear. In this study, we examined the effect of PFE on antigen-specific antibody and on cytokine production. Mice were immunized three times (weekly) with sugi basic protein (SBP), a major allergen of Japanese cedar pollen, in alum adjuvant. PFE was injected intraperitoneally into mice on day 2 before and on the day of each immunization with SBP in alum adjuvant. Serum anti-SBP IgE and IgG 1 antibody levels were significantly suppressed in mice injected with PFE. Furthermore, the production of interleukin (IL)-4, IL-5 and IL-10 by SBP-stimulated splenocyses also decreased in PFE-injected mice in a dose-dependent manner. However, PFE had no effect on either the serum anti-SBP IgG 2 a antibody levels or on interferon (INF)-gamma production by splenocytes. When splenocytes were stimulated with concanavalin A, there was no difference in cytokine production between mice injected with PFE and control mice injected with vehicle. SBP-specific T cell line established in the presence of PFE from the lymph node cells of mice immunized with SBP showed reduced IL-4, IL-5 and IL-10 production compared with that established in the absence of PFE. In contrast, comparable levels of IFN-gamma production were observed between these two T cell lines. These data suggest that PFE down-regulates Th 2-type cytokine production and prevents the Th 1/Th 2 balance from polarizing toward Th 2-type immune responses.

C-Fos-like immunoreactivity in the upper cervical spinal dorsal horn neurons following noxious chemical stimulation of the nasal mucosa in pentobarbital-anesthetized rats.

Noxious chemical stimulation of the rat nasal mucosa with mustard oil induces the expression of Fos-like immunoreactivity in trigeminal and other brain stem neurons which contribute to upper airway protective reflexes such as sneezing, coughing and apnea. To examine the role of nociceptive processing in the upper cervical spinal cord, we investigated the Fos-like immunoreactivity of the brainstem and upper cervical spinal cord following the injection of mustard oil (10 microl of 10%) into the nasal mucosa of pentobarbital anesthetized rats. Two hours after the application of mustard oil, numerous Fos-immunoreactive neurons were found in the mediolateral end of the C1 and dorsolateral division of the C2. The mean numbers of the Fos-immunoreactive neurons in the laminae I and II of the ipsilateral first and second spinal segments were significantly greater than in the control (vehicle treated) rats. There were no significant differences in the mean number of Fos-immunoreactive neurons in the contralateral C1 between the mustard oil and vehicle-treated rats. These results suggest that the C1-C2 dorsal horn neurons process the nociceptive information from nasal mucosa as well as other areas innervated by the trigeminal nerves, and that ethmoidal nerves may contribute to the exclusive conveyance of nociceptive information.

Isolation and characterization of di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins) produced by reverse action of alpha-mannosidase from jack bean.

Di- and tri-mannosyl-cyclomaltoheptaoses (beta-cyclodextrins, beta CDs), which were synthesized together with monomannosyl-beta CD in a large-scale production by reverse action of alpha-mannosidase from jack bean, were isolated and purified by HPLC. The structures of seven isomers of di-mannosyl-beta CD and six isomers of tri-mannosyl-beta CD were elucidated by FABMS and NMR spectroscopy, and by enzymatic methods.

Enzymatic synthesis, isolation, and analysis of novel alpha- and beta-galactosyl-cycloisomalto-octaoses.

Novel branched cycloisomalto-octaoses (CI8s) were enzymatically synthesized by transgalactosylation with alpha-galactosidase from coffee bean and beta-galactosidase preparations from Penicillium multicolor and Bacillus circulans, using melibiose and lactose as donor substrates, and CI8 which is a cyclic homogeneous oligosaccharide composed of eight glucose units bound by alpha-(1-->6)-linkages, as an acceptor. alpha-Galactosyl-CI8s and beta-galactosyl-CI8s obtained were isolated and purified by HPLC. Their structures were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOFMS) and NMR spectroscopy.

[Sennosides Reference Standard (Control 951) of the National Institute of Health Sciences].

The "Sennosides Reference Standard (Control 951)" was prepared, which is intended to be used for the fluorophotometric assay of sennosides content in the preparation of "Sennosides". In this assay hydroxylated mono- and dianthraquinone glucosides are chelated with boric acid, and the fluorescence intensity of the chelate is determined against that of the Reference Standard (RS). In the establishment of this RS, sennosides content in the candidate material must be determined accurately by fluorophotometry. The Sennoside AB for assay, prepared as an equimolar mixture of the purified sennoside A and Sennoside B, was used as the RS for the fluorophotometry. Based on the above concept, sennosides content in the candidate was determined as calcium salts to be 60.1 +/- 1.6% by the fluorophotometry. Thus the sennosides content of this Sennosides RS was certified to be 60%. Separately, contents of Sennoside A (SA) and Sennoside B (SB) in this candidate were determined by using HPLC. As a result, the sum of SA and SB was estimated to be 38% as free acids. Thus it was suggested that about 20% of dianthraquinone glucosides other than SA and SB and anthraquinone glucosides may be included in this Sennoside RS as free acids. Analytical results on the USP Sennosides RS were also shown and discussed, compared with the present Sennosides RS.

Isolation and characterization of novel heterogeneous branched cyclomalto-oligosaccharides (cyclodextrins) produced by transgalactosylation with alpha-galactosidase from coffee bean.

Transgalactosylated derivatives of cyclomalto-hexaose (alpha CD), -heptaose (beta CD), and -octaose (gamma CD) were synthesized by alpha-galactosidase from coffee bean using melibiose as a donor and alpha CD, beta CD or gamma CD as an acceptor. Mono- and di-O-alpha-D- galactosylated CDs were isolated and purified by HPLC. Their structures were elucidated by fast-atom bombardment mass spectrometry (FABMS) and 13C NMR spectroscopy. For structural determination of positional isomers of 6(1),6n-di-O-alpha-D-galactosyl-CDs, digestion products with cyclodextrin glucanotransferase were analyzed by HPLC and FABMS.

Galactosylation of cyclodextrins and branched cyclodextrins by alpha-galactosidases.

Transgalactosylated derivatives of cyclodextrins (CDs) and glucosyl and maltosyl CDs (G1- and G2-CDs) were synthesized by alpha-galactosidases from coffee bean and Mortierella vinacea (M. vinacea). The structures of the transfer products were analyzed by FAB-mass, 13C-NMR and methylation. Coffee bean alpha-galactosidase transferred a galactosyl residue not only to side chains of G1-CDs and G2-CDs, but also directly to CD rings. M. vinacea alpha-galactosidase transferred a galactosyl residue only to side chains of G2-CDs.

Acceptor specificities of alpha-mannosidases from jack bean and almond, and transmannosylation of branched cyclodextrins.

Jack bean alpha-mannosidase had a wide acceptor specificity and could transfer mannosyl residues to various acceptors such as D-fructose, L-arabinose, maltose, lactose, and sucrose. The structures of the transferred products of branched cyclodextrins (CDs) (glucosyl-beta CD, maltosyl-alpha CD, and maltosyl-beta CD) were found to be alpha-D-mannosyl-(1-->6)-alpha-D-glucosyl-(1-->6)-beta CD, alpha-D-mannosyl- (1-->6)-alpha-D-glucosyl-(1-->4)-alpha-D-glucosyl-(1-->6)-alpha CD and alpha-D-mannosyl-(1-->6)-alpha-D-glucosyl-(1-->4)-alpha-D-glucosyl-(1--> 6)- beta CD, respectively. Almond alpha-mannosidase also produced the same transmannosylated products of branched CDs.

Studies on lens-aldose-reductase inhibitor in medicinal plants. II. Active constituents of Monochasma savatierii Franch. et Maxim.

The 70% acetone extract of Monochasma savatierii FRANCH. et MAXIM. showed very strong inhibition of rabbit lens aldose reductase (AR). From the active fraction, five iridoid glucosides along with the two phenolic glycosides, acteoside and dehydroacteoside, have been isolated. Among them, acteoside showed the highest activity, being about 2.5 times more potent than baicalein, a known natural inhibitor of AR (IC50 = 9.8 x 10(-7) M). Demethylmussaenoside and 7-O-acetyl-8-epi-loganic acid, which are iridoid glucosides, had weak inhibitory activity.