The discovery, about forty years ago, of α-bungarotoxin, a three-finger α-neurotoxin from Bungarus multicinctus venom, enabled the isolation of the nicotinic acetylcholine receptor (nAChR), making it one of the most thoroughly characterized receptors today. Since then, the sites of interaction between α-neurotoxins and nAChRs have largely been delineated, revealing the remarkable plasticity of the three-finger toxin fold that has optimally evolved to utilize different combinations of functional groups to generate a panoply of target specificities to discern subtle differences between nAChR subtypes. New facets in toxinology have now broadened the scope for the use of α-neurotoxins in scientific discovery. For instance, the development of short, combinatorial library-derived, synthetic peptides that bind with sub-nanomolar affinity to α-bungarotoxin and prevent its interaction with muscle nAChRs has led to the in vivo neutralization of experimental α-bungarotoxin envenomation, while the successful introduction of pharmatopes bearing “α-bungarotoxin-sensitive sites” into toxin-insensitive nAChRs has permitted the use of various α-neurotoxin tags to localize and characterize new receptor subtypes. More ambitious strategies can now be envisaged for engineering rationally designed novel activities on three-finger toxin scaffolds to generate lead peptides of therapeutic value that target the nicotinic pharmacopoeia. This review details the progress made towards achieving this goal.

抄録全体を表示

The aim of this study was to investigate the effect of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole) on spontaneous contractions and levels of cyclic GMP (cGMP) of myometrial strips isolated from pregnant rats. It is a nitric oxide-independent soluble guanylate cyclase activator. Myometrial strips were obtained from eight pregnant Wistar albino rats and were mounted in organ baths for the recording of isometric tensions. We evaluated the effect of increasing concentrations of YC-1 on spontaneous myometrial contractions and on contractions of myometrial smooth muscle pretreated with methylene blue (10⁻⁵ M), tetraethylammonium chloride (TEA) (3 × 10⁻⁴ M), and glibenclamide (10⁻⁶ M). YC-1 (10⁻⁹ – 3 × 10⁻⁵ M) concentration-dependently decreased the amplitude and frequency of spontaneous contractions of myometrial strips. The inhibition of the amplitude and frequency of spontaneous contractions by YC-1 were antagonized with methylene blue (10⁻⁵ M) and TEA (3 × 10⁻⁴ M), but they were not changed by glibenclamide (10⁻⁶ M); however, the antagonistic effect of methylene blue was significantly more than that of TEA (P<0.05). We also evaluated the effect of YC-1 on the levels of cGMP in myometrial strips obtained from pregnant rat uterine horns. YC-1-stimulated myometrial strips showed an excessive elevation in myometrial cGMP that declined slowly during the subsequent washout period. These results show that YC-1 decreases spontaneous contractile activity of myometrial strips isolated from pregnant rat and causes elevation of myometrial cGMP levels in vivo. This effect of YC-1 is significantly reduced by the methylene blue and TEA, suggesting the activation of soluble guanylate cyclase and Ca²⁺-sensitive K⁺ channels as the mechanisms of action.

抄録全体を表示

To study calcium imaging data of cell populations that have various response patterns in peak amplitude and frequency of calcium oscillation in response to stimulation, comprehensive characterization based on statistical analysis of each response is important. In cultures of cells that are flat and in contact with each other, it is difficult to distinguish individual cells in calcium imaging data. We have developed a novel method to determine areas corresponding to individual cells in calcium imaging data. Rat neonatal cerebral astrocytes were filled with the calcium indicator Fura2, stained with acridine orange, and illuminated with UV light. The cell nuclei were clearly visualized. In addition, the images of these nuclei were useful for analyzing concentration-dependent alteration of calcium oscillation of cultured astrocytes in response to glutamate. This novel method may be useful for studying factors affecting calcium response patterns of cultured cell populations, including culture conditions, stimulus paradigms, and synthetic compounds.

抄録全体を表示

To elucidate the anti-inflammatory action of nicotine-induced corticosterone elevation on the passive skin Arthus reaction (PSAR), we investigated the inflammatory process in the PSAR. The polymorphonuclear leukocyte (PMNs) infiltration was observed just before as well as after elicitation by measuring extractable myeloperoxidase. The plasma exudation was significantly inhibited by anti-rat tumor necrosis factor (TNF)-α antibody (5 μg/site, i.d.) at the time of sensitization or by superoxide dismutase (52500 units/kg, i.p.) 1 h before elicitation or N^G-nitro-L-arginine-methyl ester (100 mg/kg, i.v.) just at elicitation. Pretreatment with a single injection of nicotine (0.8 mg/kg, i.p.) 30 min before elicitation suppressed the plasma exudation but not the PMNs infiltration. This nicotine-induced decreasing effect was abolished in animals supplemented with L-arginine (300 mg/kg, i.v.) just at elicitation. The production of nitric oxide (NO) in peritoneal PMNs derived from an animal injected peritoneally with oyster glycogen was significantly suppressed by pretreatment with nicotine (0.8 mg/kg, i.v.) 30 min prior to harvesting. This inhibitory action of nicotine was abolished in animals pretreated with mifepristone (30 mg/kg, s.c.), a glucocorticoid receptor antagonist. These findings indicate that a single systematic administration of nicotine may attenuate the plasma exudation in the PSAR by suppressing the production of NO in the PMNs primed with TNF-α via nicotine-induced endogenous glucocorticoid.

抄録全体を表示

This experiment was designed to test the hypothesis that endogenous corticotropin-releasing factor (CRF) contributes to the neurodegenerative process following an ischemic insult. To test this hypothesis, the effects of chronic intracerebroventricular administration of CRF or astressin, a CRF-receptor antagonist, on the decrease in the Schaffer collateral-CA1 field potential induced by hypoxia/hypoglycemia (ischemia), were tested in rat hippocampal slices. The chronic treatment with CRF had a significant exacerbating effect on the 10-min ischemia, a condition that did not affect the evoked synaptic response in the hippocampal CA1 area, as compared to vehicle-treated rats. On the other hand, astressin had a significant ameliorative effect on the 15-min ischemia-induced reduction of the evoked synaptic response in the hippocampal CA1 area. These findings suggest that CRF accelerates hippocampal ischemic vulnerability induced by hypoxia and hypoglycemia.

抄録全体を表示

To elucidate the action and signal transduction of lysophosphatidylcholine (LPC), we challenged a set of LPC on U937 human monocytes and found that LPC mobilized Ca²⁺. The Ca²⁺ response was not blocked by pertussis toxin, an inhibitor of G_i/o proteins, or by U73122, a phospholipase C inhibitor. Furthermore, the response was totally blocked by addition of EGTA to the extracellular media, suggesting that Ca²⁺ influx across the plasma membrane was the only source of LPC-induced Ca²⁺ response in the U937 cells. 16:0 and 18:0 LPC induced similar responses. Recently it has been suggested that two G protein-coupled receptors function as LPC receptors in the plasma membrane. RT-PCR analysis indicated that neither the G2A receptor nor the GPR4 receptor is expressed in the U937 monocytes. Our data suggests that another action mechanism of LPC may be involved in the LPC response in the U937 cells.

抄録全体を表示

This study investigated the estrogenic effect of Pueraria mirifica (P. mirifica) on menstrual cycle length and hormone-related ovarian function. Nine normal cyclic monkeys (Macaca fascicularis) were separated into 3 groups; each group was force fed with a single dose of 10, 100, and 1,000 mg of P. mirifica. The experimental schedule was separated into the pre-treatment and post-treatment periods. Blood samples were collected on days 3, 9 – 14, 19, 24, 29, and every 10 days until the next menstruation for one and two menstrual cycles during two consecutive periods and assayed for serum levels of gonadotropins and ovarian hormones. The result showed a significant increase in lengths of the follicular phase and total menstrual cycle in monkeys treated with 1,000 mg of P. mirifica, but no change in menstrual cycle length in monkeys treated with 10 and 100 mg of P. mirifica. Serum levels of follicle stimulating hormone, luteinizing hormone, estradiol, progesterone, or immunoreactive-inhibin did not change during the first and second menstrual cycles of the post-treatment period for all monkey groups. Our findings demonstrate that although changes in hormonal levels could not be observed in this study, a single dose of 1,000 mg of P. mirifica can disturb ovarian function and menstrual cycle in monkeys.

抄録全体を表示

Effects of ethanol extracts of three Chinese medicinal plants, namely, Qinpi (Fraxini cortex), Kushen (Sophora flavescens, AITON), and Huanglian (Coptis teeta, WALLICH), on ion transport of the rat intestinal epithelia were determined in this study. Rat intestinal epithelia mounted in an Ussing chamber attached to a voltage/current clamp were used for measuring changes in the short circuit current across the epithelia. Activation of the intestinal epithelia by serosal administration of 5 μM forskolin resulted in an increase in basal short circuit current. The ethanol extracts of each of the three plants partially reduced the current stimulated by forskolin. In the following experiments, ouabain and bumetanide were added prior to adding the ethanol extract of these plants for revealing their effect on Na⁺ and Cl⁻ movement. The results suggest that the ethanol extract of the Qinpi would affect Cl⁻ transport. On the contrary, the ethanol extract of Kushen would affect Na⁺ transport rather than Cl⁻ movement. This study provides evidences that reveal the pharmacological mechanism of the Chinese plants with anti-diarrheal properties.

抄録全体を表示

The present study was undertaken to examine whether plasma fibrinogen persistently elevates in subtotal nephrectomized rats, an animal model with inflammatory renal changes. Eight weeks after the induction of 5/6 nephrectomy in male Wistar rats, plasma fibrinogen concentration was determined for the next 12 weeks in the animals received vehicle or an angiotensin-converting enzyme inhibitor, cilazapril (1 or 10 mg/kg per day) orally. In the vehicle-treated nephrectomized rats, plasma fibrinogen concentration significantly (P<0.001) increased (from 127.3 ± 4.6 [S.E.M.] to 182.3 ± 5.2 mg/dL) compared with that in the control rats (from 118.0 ± 2.0 to 153.5 ± 5.4 mg/dL). Cilazapril attenuated the increases in plasma fibrinogen concentration in a dose-dependent manner. Serum concentration of monocyte chemoattractant protein-1, a key macrophage chemoattractant and activator, increased in the vehicle-treated nephrectomized rats, which was also reduced by cilazapril. These results suggest that plasma fibrinogen elevates persistently in the nephrectomized rats. Local inflammation may be involved in the hepatic fibrinogen synthesis in this model.

抄録全体を表示

The present study was designed to compare the effects of a selective 5-HT₃-receptor antagonist, alosetron, on the glycerol-and colorectal distention (CRD)-induced visceral nociception as measured by changes in EMG of the external oblique muscle in conscious rats. Both glycerol and CRD evoked the EMG response, and these amplified EMG were attenuated by morphine, indicating that these responses might reflect visceral nociceptive responses. In the present study, we showed that alosetron significantly attenuated the glycerol-induced visceral pain, but not that of CRD. These results suggest that the mechanism of glycerol-induced visceral nociception are apparently different from that of CRD.

抄録全体を表示

We examined possible involvement of nitric oxide synthase (NOS) on ganglion cell death in explant cultures of neonatal rat retina. Survival of retinal ganglion cells was significantly prolonged by a broad-spectrum NOS inhibitor N^ω-nitro-L-arginine methylester. NADPH diaphorase staining revealed a diffused distribution of NOS activity in neuropils of the inner plexiform layer as well as several neurons in the inner nuclear layer. Moreover, 7-nitroindazole but not aminoguanidine promoted the survival of retinal ganglion cells. These results suggest a crucial role of neuronal NOS-derived nitric oxide in retinal ganglion cell death.

抄録全体を表示

Mesangial cell (MC) proliferation, mediated by platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-β1, and cyclin-dependent kinases (CDK), is the common feature of glomerulosclerosis. Magnolia officinalis, stem bark of Machilus thunbergii S., has multiple pharmacological effects. In this study, we investigated the influence of aqueous extract of Magnolia officinalis on MC proliferation, DNA synthesis, and expression of PDGF-BB, TGF-β1, CDK1, CDK2, and CDK4 in fetal bovine serum (FBS)-activated human MC. Magnolia officinalis inhibited the MC proliferation, DNA synthesis, and the expression of PDGF-BB, CDK1, and CDK2 gene and CDK1, CDK2, and TGF-β1 protein. These results suggest that the inhibitory effect of Magnolia officinalis on MC proliferation may be mediated by regulation of PDGF-BB and TGF-β1expressions and by modulation of CDK1 and CDK2 expression.

抄録全体を表示