A new class of neurotoxin from wasp venom slows inactivation of sodium current.

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX slowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Syntheses and biological evaluation of novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues.

Novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues were efficiently synthesized and their biological activities were evaluated. 2alpha-Methyl-1alpha,25-dihydroxyvitamin D3 (2), whose unique biological activities were previously reported, was modified to 2alpha-alkyl (ethyl and propyl) and 2alpha-hydroxyalkyl (hydroxymethyl, hydroxyethyl, and hydroxypropyl) analogues 3-7 by elongation of the alkyl chain and/or introduction of a terminal hydroxyl group. 2alpha-Hydroxypropyl-1alpha,25-dihydroxyvitamin D3 (7) exhibited an exceptionally potent calcium-regulating effect and a unique activity profile.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins.

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation.

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado).

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.

Isolation and structure of pompilidotoxins, novel peptide neurotoxins in solitary wasp venoms.

Novel peptide neurotoxins, alpha- and beta-pompilidotoxins (alpha- and beta-PMTXs), were purified from the venoms of the solitary wasps Anoplius samariensis and Batozonellus maculifrons. Their structures were analyzed mostly by MALDI-TOF-MS, which were corroborated by solid-phase synthesis. alpha-PMTX, with 13 amino acid residues and the sequence of Arg-Ile-Lys-Ile-Gly-Leu-Phe-Gln-Asp-Leu-Ser-Lys-Leu-NH2, greatly potentiates synaptic transmission of lobster leg muscle by the presynaptic mechanisms. beta-PMTX, in which the lysine residue at 12 position of alpha-PMTX was replaced with arginine, was more potent than alpha-PMTX.

Alpha-pompilidotoxin (alpha-PMTX), a novel neurotoxin from the venom of a solitary wasp, facilitates transmission in the crustacean neuromuscular synapse.

A new neurotoxin, named alpha-pompilidotoxin (alpha-PMTX) has been found in the venom of the solitary wasp Anoplius safnariensis. In the neuromuscular synapse of the lobster walking leg preparation, alpha-PMTX (10-100 micro/M) caused great enhancement of both the excitatory and inhibitory postsynaptic potentials. Recordings of the excitatory post synaptic currents (EPSCs) at the synaptic sites showed that alpha-PMTX reversibly and dose-dependently potentiates EPSCs. Alpha-PMTX may act primarily on the presynaptic membrane but the mode of action of the toxin is clearly different from other known facilitatory neurotoxins, such as alpha-latrotoxin, apamin or charybdotoxin. This novel toxin will serve as a useful tool in the research field of neuroscience.

[Six patients with pneumonitis related to blended Chinese traditional medicines].

We encountered six patients with pneumonitis related to blended chinese traditional medicine (Kampo). The duration of treatment with kampo ranged from 14 to 110 days (mean: 38 days). The most common complaints were dyspnea, fever, and dry coughing. Fine crackles were heard at the bases of both lungs. Abnormal laboratory findings included high values of C-reactive protein and glutamic-oxaloacetic transaminase in all patients, lactate dehydrogenase in 5 patients, and eosinophil count in 1 patient. Chest X-ray films and CT films revealed diffuse reticulo-nodular interstitial shadows with consolidation in both lung fields in 3 patients and pleural effusion in 1 patient. Bronchoalveolar lavage was done in 4 patients; examination of the lavage fluid showed lymphocyte alveolitis, either pure or associated with neutrophilia and eosinophilia in 3 patients. Inverted CD4/CD8 lymphocyte ratios were found in 3 patients. Transbronchial lung biopsy was done in 4 patients and specimens from 3 of those 4 showed organizing pneumonitis with thickening of alveolar septa. Lymphocyte stimulation tests were positive in 4 patients. Discontinuation of the drug (2 patients) or administration of corticosteroids (4 patients) was followed by rapid improvement. Patients being treated with kampo preparations should be observed for signs and symptoms of drug-induced pneumonitis.

Establishment of an in vitro assay system for screening hepatitis C virus protease inhibitors using high performance liquid chromatography.

The hepatitis C virus (HCV) genome contains the code for a conserved, serine-type protease, called NS3, for the processing of the non-structural protein region of the viral polyproteins. Furthermore, a related protein, NS4A, is an effector or cofactor of NS3 protease activity in the cleavage of NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. To establish an in vitro assay system for the screening of those enzyme inhibitors that inhibit the protease NS3-4A, we prepared a maltose-binding protein-NS3-NS4A fusion protein and a synthetic peptide substrate that mimics the NS5A-5B junction. Cleavage of the synthetic peptide was analyzed by reversed-phase high performance liquid chromatography (HPLC). We showed that the enzymatic activity of the NS3-NS4A fusion protein was enhanced in comparison to the NS3 protein alone. The assay conditions for optimum NS3-4A protease activity were determined to be pH 7.6 and 37 degrees C. In addition, we evaluated several protease inhibitors using the same HPLC assay system. The activity of HCV protease NS3-4A was inhibited by 2714.4 microM diisopropyl fluorophosphate, 270.8 microM N-tosyl-L-lysyl chloromethyl ketone, and 825.5 microM chymostatin. The results of the present study indicated that the synthetic peptide substrate and HPLC assay system are suitable for studying HCV protease activity and may facilitate the development of anti-HCV therapeutic reagents.

Zizyphi fructus, a constituent of antiasthmatic herbal medicine, stimulates airway epithelial ciliary motility through nitric oxide generation.

The effects of Zizyphi fructus, a major constituent of Chinese anti-asthmatic herbal medicine, on ciliary motility and nitric oxide (NO) generation in canine cultured tracheal epithelium were studied by the microphoto-oscillation method and the specific amperometric method, respectively. Addition of Zizyphi fructus at 100 micrograms/mL rapidly increased ciliary beat frequency (CBF), an effect that was inhibited by NG-nitro-L-arginine methylester (L-NAME) but not by NG-nitro-D-arginine methylester (D-NAME), and this inhibition was reversed by L-arginine but not by D-arginine. Immersion of the NO-selective electrode in the medium bathing tracheal epithelial cells showed a baseline current of 21.3-60.4 pA, which corresponded to NO concentration ([NO]) at 34.4 +/- 7.8 nM. Zizyphi fructus caused a concentration-dependent increase in [NO], the maximal increase from the baseline [NO] level and the concentration of Zizyphi fructus required to produce a half-maximal effect (EC50) being 107 +/- 14 nM (p < .001) and 7.2 +/- 2.9 micrograms/mL, respectively. These results suggest that Zizyphi fructus enhances airway ciliary motility and that this effect is exerted through the stimulation of epithelial NO generation.

Colorimetric assay system for screening antiviral compounds against hepatitis B virus.

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 microliters of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.

Effect of saiboku-to, an antiasthmatic herbal medicine, on nitric oxide generation from cultured canine airway epithelial cells.

The effect of Saiboku-to (TJ-96), an antiasthmatic Kampo medicine, on the generation of nitric oxide (NO) from cultured canine tracheal epithelium was investigated using a highly specific amperometric sensor for this molecule in vitro. Immersion of the NO-selective electrode in the medium containing tracheal epithelial cells detected the baseline current of 16.8-57.0 pA, which corresponded to an NO concentration ([NO]) of 39.7 +/- 8.1 nM. Addition of TJ-96 increased [NO] in a concentration-dependent manner, the maximal increase from the baseline level and the concentration of TJ-96 required to produce a half-maximal effect (EC50) being 127.5 +/- 20.1 nM (P < 0.001) and 86 +/- 9 micrograms/ml, respectively. Pretreatment of cells with NG-nitro-L-arginine methylester (L-NAME) greatly inhibited the TJ-96-induced increase in [NO], whereas NG-nitro-D-arginine methylester (D-NAME) had no effect, and this inhibition was reversed by L-arginine but not by D-arginine. Cytochemical staining of the epithelial cells showed marked reactivity of NADPH diaphorase activity. These results suggest that NO is spontaneously released by the airway epithelium and that TJ-96 stimulates the epithelial NO generation.

A screening system for antiviral compounds against herpes simplex virus type 1 using the MTT method with L929 cells.

We developed the improved MTT assay system for in vitro evaluation of anti-HSV-1 agents using L929 cells derived from mouse connective tissue. This assay system provides results in 4 days using a 96-well microplate. The EC50 values of several anti-HSV agents (ACV, BVaraU, and others) were found to be similar to those obtained by the plaque reduction method using MRC-5 cells. The present MTT assay is rapid, accurate, and may be useful as an automatic screening system for evaluation of anti-HSV-1 compounds.

Effect of TJ-96, an anti-allergic herbal medicine, on tracheal transepithelial potential difference in vivo.

We studied the effect of Saiboku-to (TJ-96), an anti-allergic herbal medicine, on transepithelial potential difference of rabbit trachea and possible involvement of nitric oxide (NO) generation in vivo. Perfusion of TJ-96 on the tracheal mucosal surface increased PD in a concentration-dependent manner, the maximal increase from the baseline level and the concentration of TJ-96 required to produce a half-maximal effect (EC50) being 8.1 +/- 1.4 mV (mean +/- SE, P < 0.001) and 47 micrograms/ml. This effect was abolished by pretreatment with the Na channel blocker amiloride. NG-nitro-L-arginine methylester (L-NAME) but not NG-nitro-D-arginine methylester (D-NAME) inhibited TJ-96-induced increase in PD, and this inhibition was selectively reversed by L-arginine. These results suggest that TJ-96 stimulates Na absorption by airway epithelial cells probably through NO generation.

[Effect of Sei-hai-to on ion transport in airway epithelial cells].

The effect of Sei-hai-to on ion transport in airway epithelial cells and its mechanism of action were investigated. Measurement of short-circuit current (Isc) using cultured epithelial cells from canine tracheal mucosa under short-circuit conditions showed that Isc was dose-dependently increased by the submucosal administration of Sei-hai-to. The response of Isc to this drug was not affected by pretreatment the cells with amiloride, but abolished by furosemide, or Cl-free condition. These results suggest that Sei-hai-to stimulates Cl ion transport selectively and may affect the subsequent movement of water across the airway mucosa to the lumen.

Effect of unilateral motor cortex ablation on activity of choline acetyltransferase and levels of amino acid transmitter candidates in the spinal cord of adult monkeys.

Evidence that L-glutamate is a neurotransmitter of corticofugal fibers was sought by measuring changes in several biochemical markers of neurotransmitter function in discrete regions of spinal cord after ablation of sensorimotor cortex in monkeys. One and five weeks after unilateral cortical ablation, samples from six areas of spinal cord (ventral, lateral and dorsal regions of the left and right sides) were analysed for choline acetyltransferase (ChAT) activity and contents of amino acid transmitter candidates-glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), taurine (Tau) and gamma-aminobutyric acid (GABA). During one to five weeks after unilateral cortical ablation of the monkey, prolonged hemiplegia in the contralateral side was observed. Histological examination of the spinal cord 5 weeks after unilateral (left) cortical ablation showed no apparent change in either control (ipsilateral, left) or affected (contralateral, right) sides of the cord as examined by the Klüver-Barrera method. The ChAT activity as a cholinergic marker was scarcely changed in any region of either left (control) or right (affected) side of the spinal cord at one and five weeks after unilateral (left side) ablation of the motor cortex. Amino acid levels in each region of the spinal cord were not significantly changed one week after unilateral ablation of the motor cortex. However, a significant decrease of Glu content was observed in the lateral column of the affected (right) side compared to the control (left) side of cervical and lumbar cord five weeks after cortical ablation of the left motor area. No concomitant alterations of other amino acids were detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiation of beta-adrenergic function by saiboku-to and bakumondo-to in canine bronchial smooth muscle.

To determine the effects of the Kampo drugs Saiboku-to, Bakumondo-to and Orengedoku-to on beta-adrenergic function in airway smooth muscle, we studied isolated canine bronchial segments under isometric conditions in vitro. Incubation of tissues with these drugs did not alter the contractile responses to acetylcholine and histamine. The relaxation of tissues precontracted with acetylcholine induced by isoproterenol was augmented by Saiboku-to and Bakumondo-to, so that the concentration of isoproterenol required to produce a half-maximal effect (IC50) was decreased from 4.6 +/- 0.5 x 10(-8) M to 1.9 +/- 1.0 x 10(-8) M (P < 0.05) and to 1.0 +/- 0.8 x 10(-8) M (P < 0.01), respectively, whereas Orengedoku-to had no effect. These effects were concentration-dependent with the threshold concentrations being 0.3 mg/ml for Saiboku-to and 0.1 mg/ml for Bakumondo-to. Saiboku-to and Bakumondo-to did not affect cyclic AMP levels in airway smooth muscle per se but potentiated the isoproterenol-induced cyclic AMP accumulation. These results suggest that Saiboku-to and Bakumondo-to but not Orengedoku-to potentiate beta-adrenergic function in airway smooth muscle, which may reflect the efficacy of these drugs on airway hyperresponsiveness and asthma.

Angiotensin II-1 receptor-mediated Cl secretion by canine tracheal epithelium.

To elucidate the effect of angiotensin II (AII) on ion transport function of airway epithelium, we studied the bioelectrical properties of canine cultured tracheal epithelium under short-circuit conditions in vitro. Addition of AII to submucosal solution in Ussing chambers increased the short-circuit current (ISC) in a dose-dependent fashion, the maximal increase from the baseline value and the concentration required to produce a half-maximal effect being 5.2 +/- 0.5 microA/cm2 (p < 0.001) and 10(-6) M, respectively. In contrast, mucosal AII had little effect. The AII-induced increase in ISC was not altered by the AII-2 receptor antagonist EXP655 but was depressed by the AII-1 receptor antagonist DuP 753. Diphenylamine-2-carboxylate, Cl-free medium, indomethacin, the phospholipase A2 inhibitor mepacrine, and the methyltransferase inhibitor 3-deazaadenosine reduced the change in ISC, whereas amiloride and the lipoxygenase inhibitor AA-861 did not. Addition of AII to the submucosal but not the mucosal side increased the release of prostaglandin E2, an effect that was abolished by DuP 753. These results suggest that AII may interact with the submucosal AII-1 receptor and stimulate Cl secretion across tracheal epithelium through the mobilization of arachidonic acid and the release of prostaglandin E2.

[Effect of saibokuto on mucociliary transport system in the airway--basic and clinical assessments].

We studied the effects of Saibokuto, an antiasthmatic agent, on the airway mucociliary transport system. The addition of Saibokuto to a Rose chamber containing rabbit cultured tracheal epithelium dose-dependently increased ciliary beat frequency (CBF) as assessed by a photoelectric method: the maximal increase from the baseline value was 31.5 +/- 5.2% (p less than 0.001) and the concentration of Saibokuto required to produce a half-maximal effect (EC50) was 10(-4) mg/ml. This effect was not affected by the beta-adrenergic receptor antagonist propranolol or Ca(2+)-free medium, but was inhibited by the cyclooxygenase inhibitor indomethacin. Intracellular cyclic AMP levels were increased from 45.6 +/- 8.2 to 72.1 +/- 12.6 pmole/mg protein by 1.0 mg/ml Saibokuto (p less than 0.05). Thus, Saibokuto enhances ciliary motility probably through the synthesis of cyclooxygenase products and cyclic AMP. Additionally, administration of this drug to patients with chronic bronchitis, asthma and bronchiectasis improved their problems with sputum expectoration and reduced the amount of daily sputum (p less than 0.001). Therefore, Saibokuto may be of value in the treatment of patients with impaired mucociliary transport function in the airway.

Stimulation of Na absorption by the antiasthmatic kampo drug Saiboku-to in cultured airway epithelium.

To study the effect of the Kampo drug Saiboku-to (TJ-96) on ion transport function of airway epithelial cells, we studied bioelectric properties of cultured tracheal epithelium from dogs under short-circuit conditions in vitro. Addition of TJ-96 (1 mg/ml) to the mucosal solution of the Ussing chamber increased the epithelial short-circuit current (SCC) from 6.5 +/- 0.7 to 11.4 +/- 1.6 microA/cm2 (P less than 0.001). This effect was dose-dependent, with the maximal increase from the baseline value and the concentration required to produce a half-maximal effect (EC50) being 70.5 +/- 12.6% (P less than 0.001) and 3 micrograms/ml, respectively; and there were corresponding increases in transepithelial potential difference and cell conductance. Submucosal addition of TJ-96 likewise increased SCC, although the magnitude of the response was smaller as compared with the response to the mucosal addition. The TJ-96-induced increase in SCC was not affected by diphenylamine-2-carboxylate or furosemide but abolished by amiloride. Intracellular cyclic AMP levels were dose-dependently increased by TJ-96. These results indicate that TJ-96 may selectively stimulate Na absorption across the tracheal epithelium, probably through intracellular accumulation of cyclic AMP.